Anti PD-1 antibodies

ABSTRACT

The present invention relates to antibodies and antigen-binding fragments thereof that bind to PD-1, and to methods of using such antibodies and antigen-binding fragments. For example, the present invention provides humanized anti-PD-1 antibodies and methods of use thereof.

RELATED APPLICATIONS

This application is a Continuation which claims benefits of U.S.application Ser. No. 16/538,448 filed Aug. 12, 2019, which is aDivisional of U.S. application Ser. No. 15/328,225 filed Jan. 23, 2017,now issued as U.S. Pat. No. 10,428,146, which is a U.S. National Stageof International Application No. PCT/US2015/041575, filed Jul. 22, 2015,which claims priority to International Application No.PCT/CN2014/082721, filed Jul. 22, 2014, which is incorporated herein byreference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to antibodies and antigen-bindingfragments thereof that bind to PD-1, and to methods of using suchantibodies and antigen-binding fragments.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The content of the text file submitted electronically herewith isincorporated herein by reference in its entirety: A computer readableformat copy of the Sequence Listing (filename:CBTH_006_03US_SeqList_ST25); date recorded: Mar. 8, 2021; file size 147KB).

BACKGROUND

Programmed death receptor 1 (PD-1) is primarily expressed on lymphocytesand has two ligands, PD-L1 and PD-L2. PD-1 is a 55 kDa protein encodedby a gene Pdcd1 and was shown to down-regulate antigen receptorsignaling driven by its ligand's engagement (Freeman et al. (2000) J ExpMed 192:1027-34; Latchman, et. al. (2001) Nat Immunol 2:261-8; Carter etal. (2002) Eur J Immunol 32:634-43). PD-1 belongs to the immunoglobulinsuperfamily which includes members such as CD28, CTLA-4, ICOS and BTLA.PD-1 is type I transmembrane glycoprotein containing an Ig variable-type(V-type) domain for ligand binding and a cytoplasmic tail for thebinding of signaling molecules. PD-1 contains two cytoplasmictyrosine-based signaling motifs, an immunoreceptor tyrosine-basedinhibition motif (ITIM) and an immunoreceptor tyrosine-based switchmotif (ITSM). Following T cell stimulation, PD-1 recruits the tyrosinephosphatase SHP-2 to the ITSM motif within its cytoplasmic tail, leadingto the dephosphorylation of effector molecules such as CD3 Zeta, PKCtheta and ZAP70 that are involved in the CD3 T cell signaling cascade.In contrast, PD-1's ligands (PD-L1 and PD-L2) have two short cytoplasmicregions with no known functions. The ligands have an extracellularregion containing IgV- and IgC-like domains and are constitutivelyexpressed or can be induced in a variety of cell types, includingnon-hematopoietic tissues as well as various tumor types. PD-L1 is notonly expressed on B, T, myeloid and dendritic cells (DCs), but also onperipheral cells, like microvascular endothelial cells and non-lymphoidorgans like heart, lung etc. In contrast, PD-L2 is only found onmacrophages and DCs. The expression pattern of PD-1 ligands issuggestive of a role for PD-1 in maintaining peripheral tolerance andmay serve regulate self-reactive T- and B-cell responses in theperiphery. To date, numerous studies have shown that interaction of PD-1with its ligands leads to the inhibition of lymphocyte proliferation invitro and in vivo. Disruption of the PD-1/PDL1 interaction has beenshown to increase T cell proliferation and promote cytokine production.

Thus, there is an important role for the PD-1/PD-L1 pathway incontrolling immune responses. Dysfunction of PD-1/PD-L1 signalingappears to be correlated with initiation and development of diseasessuch as cancer and viral infection. Analysis of knockout animals has ledto the understanding that PD-1 functions mainly in inducing andregulating peripheral tolerance. Thus, therapeutic blockade of the PD-1pathway would be helpful in overcoming immune tolerance and in thetreatment of cancer or infection as well as in boosting immunity duringvaccination (either prophylactic or therapeutic). There is a need in theart for improved methods for blocking the PD-1 pathway.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides antibodies andantigen-binding fragments thereof that bind to programmed death receptor1 (PD-1). In some embodiments, the antibodies and antigen-bindingfragments thereof bind to human PD-1. In some embodiments, theantibodies and antigen-binding fragments thereof bind to PD-1 and blockbinding of PD-L1 and/or PD-L2 to PD-1. In further embodiments, theanti-PD-1 antibodies and fragments thereof bind to PD-1 and disrupt thePD-1/PD-L1 or PD1/PD-L2 pathway. In one embodiment, the antibody orfragment thereof is a murine antibody, a chimeric antibody, a humanantibody or a humanized antibody. In one embodiment, the anti-PD-1antibody or fragment thereof is a monoclonal antibody, scFv, Fabfragment, Fab′ fragment, F(ab)′ fragment, bispecific antibody,immunoconjugate, or a combination thereof.

In one embodiment, the present invention provides an isolated antibodyor fragment thereof comprising one or more CDRs selected from the groupconsisting of SEQ ID NOs: 19-21, 24-26, 29-31, 34-36, 40-42, 45-47,50-52, 55-57, 60-62, 65-67, 70-72, 75-77, 80-82, 85-87, 90-92, 95-97,100-102, 105-107, 110-112, and 115-117.

In one embodiment, the antibody or fragment thereof comprises a lightchain CDR1 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:24, 34, 45, 55, 65, 75, 85, 95, 105, and 115.

In one embodiment, the antibody or fragment thereof comprises a lightchain CDR2 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:25, 35, 46, 56, 66, 76, 86, 96, 106, and 116.

In one embodiment, the antibody or fragment thereof comprises a lightchain CDR3 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:26, 36, 47, 57, 67, 77, 87, 97, 107, and 117.

In one embodiment, the antibody or fragment thereof comprises a heavychain CDR1 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:19, 29, 40, 50, 60, 70, 80, 90, 100, and 110.

In one embodiment, the antibody or fragment thereof comprises a heavychain CDR2 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:20, 30, 41, 51, 61, 71, 81, 91, 101, and 111.

In one embodiment, the antibody or fragment thereof comprises a heavychain CDR3 sequence having at least 80% homology, at least 81% homology,at least 82% homology, at least 83% homology, at least 84% homology, atleast 85% homology, at least 86% homology, at least 87% homology, atleast 88% homology, at least 89% homology, at least 90% homology, atleast 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toan amino acid sequence selected from the group consisting of SEQ ID NOs:21, 31, 42, 52, 62, 72, 82, 92, 102, and 112. In one embodiment, theantibody or fragment thereof comprises a light chain CDR1 consisting ofan amino acid sequence selected from the group consisting of SEQ ID NOs:24, 34, 45, 55, 65, 75, 85, 95, 105, and 115; a light chain CDR2consisting of an amino acid sequence selected from the group consistingof SEQ ID NOs: 25, 35, 46, 56, 66, 76, 86, 96, 106, and 116; a lightchain CDR3 consisting of an amino acid sequences selected from the groupconsisting of SEQ ID NOs: 26, 36, 47, 57, 67, 77, 87, 97, 107, and 117;a heavy chain CDR1 consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 19, 29, 40, 50, 60, 70, 80, 90, 100,and 110; a heavy chain CDR2 consisting of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 20, 30, 41, 51, 61,71, 81, 91, 101, and 111 and a heavy chain CDR3 consisting of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 21, 31,42, 52, 62, 72, 82, 92, 102, and 112.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 24, 25, and26, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:19, 20, and 21, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a light chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 24, 25, and 26, respectively, and a heavy chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 19, 20, and 21,respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 34, 35, and36, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:29, 30, and 31, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a light chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 34, 35, and 36, respectively, and a heavy chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 29, 30, and 31,respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 45, 46, and47, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:40, 41, and 42, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a light chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 45, 46, and 47, respectively, and a heavy chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 40, 41, and 42,respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 55, 56, and57, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:50, 51, and 52, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a light chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 55, 56, and 57, respectively, and a heavy chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 50, 51, and 52,respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 65, 66, and67, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:60, 61, and 62, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a light chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 65, 66, and 67, respectively, and a heavy chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 60, 61, and 62,respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 75, 76, and77, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:70, 71, and 72, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a light chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 75, 76, and 77, respectively, and a heavy chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 70, 71, and 72,respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 85, 86, and87, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:80, 81, and 82, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a light chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 85, 86, and 87, respectively, and a heavy chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 80, 81, and 82,respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 95, 96, and97, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence according to SEQ ID NOs:90, 91, and 92, respectively. In a further embodiment, the antibody orantibody fragment thereof comprises a light chain CDR1, CDR2, and CDR3according to SEQ ID NOs: 95, 96, and 97, respectively, and a heavy chainCDR1, CDR2, and CDR3 according to SEQ ID NOs: 90, 91, and 92,respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 105, 106,and 107, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence accordingto SEQ ID NOs: 100, 101, and 102, respectively. In a further embodiment,the antibody or antibody fragment thereof comprises a light chain CDR1,CDR2, and CDR3 according to SEQ ID NOs: 105, 106, and 107, respectively,and a heavy chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 100,101, and 102, respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain CDR1, CDR2, and CDR3 comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence according to SEQ ID NOs: 115, 116,and 117, respectively; and a heavy chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence accordingto SEQ ID NOs: 110, 111, and 112, respectively. In a further embodiment,the antibody or antibody fragment thereof comprises a light chain CDR1,CDR2, and CDR3 according to SEQ ID NOs: 115, 116, 117, respectively, anda heavy chain CDR1, CDR2, and CDR3 according to SEQ ID NOs: 110, 111,and 112, respectively.

In one embodiment, the antibody or fragment thereof binds PD-1 andcomprises a light chain variable region comprising an amino acidsequence having at least 80% homology, at least 85% homology, at least90% homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 23, 33, 44, 54, 64, 74, 84, 94, 104, 114, 133, 143, and 152;and a heavy chain variable region comprising an amino acid sequencehaving at least 80% homology, at least 85% homology, at least 90%homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 18, 28, 39, 49, 59, 69, 79, 89, 99, 109, 131, and 141. In afurther embodiment, the isolated antibody or fragment thereof binds PD-1and comprises a light chain variable region comprising, consistingessentially of, or consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 23, 33, 44, 54, 64, 74, 84, 94, 104,114, 133, 143, and 152; and a heavy chain variable region comprising,consisting essentially of, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 18, 28, 39, 49, 59,69, 79, 89, 99, 109, 131, and 141.

In one embodiment, the invention provides anti-PD-1 antibodies thatcomprise a variable light chain of an antibody selected from the groupconsisting of 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, 7A4,and 7A4D and a variable heavy chain of an antibody selected from thegroup consisting of 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8,and 7A4. Thus, in one embodiment, the invention provides an antibody orfragment thereof comprising a light chain variable region comprising SEQID NO: 23 and a heavy chain variable region comprising SEQ ID NO: 18; alight chain variable region comprising SEQ ID NO: 33 and a heavy chainvariable region comprising SEQ ID NO: 28; a light chain variable regioncomprising SEQ ID NO: 44 and a heavy chain variable region comprisingSEQ ID NO: 39; a light chain variable region comprising SEQ ID NO: 54and a heavy chain variable region comprising SEQ ID NO: 49; a lightchain variable region comprising SEQ ID NO: 64 and a heavy chainvariable region comprising SEQ ID NO: 59; a light chain variable regioncomprising SEQ ID NO: 74 and a heavy chain variable region comprisingSEQ ID NO: 69; a light chain variable region comprising SEQ ID NO: 84and a heavy chain variable region comprising SEQ ID NO: 79; a lightchain variable region comprising SEQ ID NO: 94 and a heavy chainvariable region comprising SEQ ID NO: 89; a light chain variable regioncomprising SEQ ID NO: 104 and a heavy chain variable region comprisingSEQ ID NO: 99; a light chain variable region comprising SEQ ID NO: 114and a heavy chain variable region comprising SEQ ID NO: 109; a lightchain variable region comprising SEQ ID NO: 133 and a heavy chainvariable region comprising SEQ ID NO: 131; a light chain variable regioncomprising SEQ ID NO: 143 and a heavy chain variable region comprisingSEQ ID NO: 141; or a light chain variable region comprising SEQ ID NO:152 and a heavy chain variable region comprising SEQ ID NO: 131.

In one embodiment, the present invention provides a chimeric anti-PD-1antibody, wherein the antibody comprises a heavy chain having an aminoacid sequence having at least 80% homology, at least 85% homology, atleast 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs; 119, 121, 125, and 127; and a light chainhaving an amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 123 and 129.

In one embodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a heavy chain variable regionhaving an amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 131 and 141. In anotherembodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a light chain variable regionhaving an amino acid sequence having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 133, 143 and 152.

In another embodiment, the present invention provides a humanizedanti-PD-1 antibody, wherein the antibody comprises a heavy chainvariable region having at least 80% homology, at least 85% homology, atleast 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to SEQ ID NO: 131 and a light chain variable regionhaving least 80% homology, at least 85% homology, at least 90% homology,at least 91% homology, at least 92% homology, at least 93% homology, atleast 94% homology, at least 95% homology, at least 96% homology, atleast 97% homology, at least 98% homology, or at least 99% homology toSEQ ID NO: 133 or 152. In another embodiment, the present inventionprovides a humanized anti-PD-1 antibody, wherein the antibody comprisesa heavy chain variable region having at least 80% homology, at least 85%homology, at least 90% homology, at least 91% homology, at least 92%homology, at least 93% homology, at least 94% homology, at least 95%homology, at least 96% homology, at least 97% homology, at least 98%homology, or at least 99% homology to SEQ ID NO: 141 and a light chainvariable region having least 80% homology, at least 85% homology, atleast 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to SEQ ID NO: 143.

In one embodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a full heavy chain having anamino acid sequence having at least 80% homology, at least 85% homology,at least 90% homology, at least 91% homology, at least 92% homology, atleast 93% homology, at least 94% homology, at least 95% homology, atleast 96% homology, at least 97% homology, at least 98% homology, or atleast 99% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 135, 137, 145, and 147. In another embodiment,the present invention provides a humanized anti-PD-1 antibody, whereinthe antibody comprises a full light chain having an amino acid sequencehaving at least 80% homology, at least 85% homology, at least 90%homology, at least 91% homology, at least 92% homology, at least 93%homology, at least 94% homology, at least 95% homology, at least 96%homology, at least 97% homology, at least 98% homology, or at least 99%homology to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 139, 149, and 153.

In one embodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 135 and a light chain according to SEQ ID NO: 139. In anotherembodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 137 and a light chain according to SEQ ID NO: 139. In anotherembodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 135 and a light chain according to SEQ ID NO: 153. In anotherembodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 137 and a light chain according to SEQ ID NO: 153. In anotherembodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 145 and a light chain according to SEQ ID NO: 149. In anotherembodiment, the present invention provides a humanized anti-PD-1antibody, wherein the antibody comprises a heavy chain according to SEQID NO: 147 and a light chain according to SEQ ID NO: 149. In oneembodiment, the present invention provides anti-PD-1 antibodies orfragments thereof that bind to the same epitope on PD-1 as any of theexemplary antibodies provided herein. In one embodiment, the antibodiesor fragments thereof compete with any of the exemplary antibodiesprovided herein for binding to PD-1. Binding to PD-1 may be measured byELISA, flow cytometry, surface plasmon resonance (SPR) assay, or anyother method known in the art.

In one embodiment, the present invention provides anti-PD-1 antibodiesand fragments thereof that bind to PD-1 with an affinity of about 1 nMto about 0.01 nM. In a further embodiment, the anti-PD-1 antibodies andfragments thereof provided herein bind to PD-1 with an affinity of fromabout 0.5 nM to about 0.1 nM. In another embodiment, the anti-PD-1antibodies and fragments thereof provided herein bind to PD-1 with anaffinity of about 1 nM or less. In a further embodiment, the anti-PD-1antibodies and fragments thereof provided herein bind to PD-1 with anaffinity of about 0.75 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.5 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.25 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.2 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.15 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.1 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.075 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.05 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.025 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.02 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.015 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.01 nM or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.0075 or less. In a further embodiment, theanti-PD-1 antibodies and fragments thereof provided herein bind to PD-1with an affinity of about 0.005 or less.

In one embodiment, the anti PD-1 antibodies and fragments thereofprovided herein have a binding EC50 for PD-1 of about 1 ng/mL to about2000 ng/mL. In a further embodiment, the anti PD-1 antibodies andfragments thereof provided herein have a binding EC50 for PD-1 of about1 ng/mL to about 1500 ng/mL. In a further embodiment, the anti PD-1antibodies and fragments thereof provided herein have a binding EC50 forPD-1 of about 1 ng/mL to about 1000 ng/mL. In a further embodiment, theanti PD-1 antibodies and fragments thereof provided herein have abinding EC50 for PD-1 of about 2 ng/mL to about 500 ng/mL. In a furtherembodiment, the anti PD-1 antibodies and fragments thereof providedherein have a binding EC50 for PD-1 of about 2 ng/mL to about 200 ng/mL.In a further embodiment, the anti PD-1 antibodies and fragments thereofprovided herein have a binding EC50 for PD-1 of about 5 ng/mL to about100 ng/mL. In a further embodiment, the anti PD-1 antibodies andfragments thereof provided herein have a binding EC50 for PD-1 of about5 ng/mL to about 50 ng/mL. In one embodiment, the anti PD-1 antibodiesand fragments thereof provided herein have a binding EC50 for PD-1 ofabout 500 ng/mL or less, about 400 ng/mL or less, about 300 ng/mL orless, about 250 ng/mL or less, about 200 ng/mL or less, about 150 ng/mLor less, about 100 ng/mL or less, about 75 ng/mL or less, about 60 ng/mLor less, about 50 ng/mL or less, about 40 ng/mL or less, or about 30ng/mL or less.

In one embodiment, the anti PD-1 antibodies and fragments thereofprovided herein inhibit PD-L1 binding with an IC50 of about of about 1ng/mL to about 1000 ng/mL. In a further embodiment, the anti PD-1antibodies and fragments thereof provided herein inhibit PD-L1 bindingwith an IC50 of about 2 ng/mL to about 800 ng/mL. In a furtherembodiment, the anti PD-1 antibodies and fragments thereof providedherein inhibit PD-L1 binding with an IC50 of about 5 ng/mL to about 500ng/mL. In a further embodiment, the anti PD-1 antibodies and fragmentsthereof provided herein inhibit PD-L1 binding with an IC50 of about 5ng/mL to about 100 ng/mL. In a further embodiment, the anti PD-1antibodies and fragments thereof provided herein inhibit PD-L1 bindingwith an IC50 of about 10 ng/mL to about 50 ng/mL. In one embodiment, theanti PD-1 antibodies and fragments thereof provided herein inhibit PD-L1binding with an IC50 of about 800 ng/mL or less, about 400 ng/mL orless, about 300 ng/mL or less, about 250 ng/mL or less, about 200 ng/mLor less, about 150 ng/mL or less, about 100 ng/mL or less, about 75ng/mL or less, about 60 ng/mL or less, about 50 ng/mL or less, about 40ng/mL or less, or about 30 ng/mL or less.

In one embodiment, the anti-PD-1 antibody provided herein is a humanizedantibody having a light chain variable region amino acid sequenceaccording to SEQ ID NO: 133 and a heavy chain variable region amino acidaccording to SEQ ID NO: 131; or having a light chain variable regionamino acid sequence according to SEQ ID NO: 143 and a heavy chainvariable region amino acid sequence according to SEQ ID NO: 141; orhaving a light chain variable region amino acid sequence according toSEQ ID NO: 152 and a heavy chain variable region amino acid sequenceaccording to SEQ ID NO: 131; wherein the anti-PD-1 antibody has a PD-1binding EC50 of about 200 ng/ml or less or about 150 ng/mL or less orabout 100 ng/mL or less or about 80 ng/ml or less or about 60 ng/mL orless, as measured by ELISA or FACS. In another embodiment, the anti-PD-1antibody provided herein is a humanized antibody having a light chainvariable region amino acid sequence according to SEQ ID NO: 133 and aheavy chain variable region amino acid according to SEQ ID NO: 131; orhaving a light chain variable region amino acid sequence according toSEQ ID NO: 143 and a heavy chain variable region amino acid sequenceaccording to SEQ ID NO: 141; or having a light chain variable regionamino acid sequence according to SEQ ID NO: 152 and a heavy chainvariable region amino acid sequence according to SEQ ID NO: 131; whereinthe anti-PD-1 antibody has a PD-L1 blockage IC50 of about 1000 ng/mL orless, or about 800 ng/mL or less, or about 600 ng/mL or less, or about500 ng/mL or less, or about 400 ng/mL or less, or about 300 ng/mL orless, or about 200 ng/mL or less, or about 100 ng/mL or less, or about60 ng/mL or less, or about 30 ng/mL or less, or about 25 ng/mL or less,or about 20 ng/mL or less, or about 10 ng/mL or less, as measured byELISA or FACS. In another embodiment, the anti-PD-1 antibody providedherein is a humanized antibody having a light chain variable regionamino acid sequence according to SEQ ID NO: 133 and a heavy chainvariable region amino acid according to SEQ ID NO: 131; or having alight chain variable region amino acid sequence according to SEQ ID NO:143 and a heavy chain variable region amino acid sequence according toSEQ ID NO: 141; or having a light chain variable region amino acidsequence according to SEQ ID NO: 152 and a heavy chain variable regionamino acid sequence according to SEQ ID NO: 131; wherein the anti-PD-1antibody has an affinity for PD-1 of about 1 nM or less, or about 0.5 nMor less, or about 0.1 nM or less, or about 0.05 nM or less. In aparticular embodiment, the humanized anti-PD-1 antibody has an affinityfor PD-1 of about 0.1 nM.

In one embodiment, the anti-PD-1 antibodies and fragments thereofprovided bind to PD-1 on T cells, disrupting the PD-1/PD-L1 interactionand resulting in an increase in T cell activation. In a furtherembodiment, the antibodies and fragments thereof bind PD-1 and result inan increase in T cell proliferation and/or cytokine production. In a yetfurther embodiment, the antibodies and fragments thereof bind PD-1 andresult in an increase of one or more cytokines selected from the groupconsisting of IL-2, IFNγ, TNF, IL-1, IL-4, IL-5, IL-6, IL-12, IL-13,IL-17, and GM-CSF. Thus, in one aspect, the present invention providesmethods for modulating an immune response comprising contacting T cellswith the anti-PD-1 antibody or fragment thereof. In one embodiment, themodulation of an immune response by the anti-PD-1 antibodies andfragments provided herein may be measured in a mixed lymphocyte (MLR)reaction. In one embodiment, the anti-PD-1 antibodies provided hereinincrease the level of cytokine production from lymphocytes in an MLR. Ina further embodiment, the anti-PD-1 antibodies increase the level ofIL-2 production and/or IFNγ production in an MLR. In a yet furtherembodiment, the anti-PD-1 antibodies increase the level of IL-2production and IFNγ production in an MLR. In one embodiment, theanti-PD-1 antibodies enhance memory T cell responses. In a furtherembodiment, the anti-PD-1 antibodies enhance memory T cell responses asmeasured by an increase in IFNγ production from memory T cells.

In one embodiment, the anti-PD-1 antibodies and fragments thereofprovided herein inhibit regulatory T cell function. In a furtherembodiment, the anti-PD-1 antibodies and fragments thereof inhibit thesuppression of effector T cells by regulatory T cells. In anotherembodiment, the anti-PD-1 antibodies and fragments thereof restore theeffector functions of T cells in the presence of regulatory T cells. Ina further embodiment, the anti-PD-1 antibodies and fragments thereofrestore the ability of effector T cells to proliferate and/or producecytokines in the presence of regulatory T cells. Thus, in oneembodiment, the present invention provides a method for inhibiting thesuppressive effects of regulatory T cells in vitro or in a subject inneed thereof.

In one aspect, an isolated antibody or fragment thereof that binds toPD-1 is provided, wherein the antibody is produced by a hybridomaselected from the group consisting of the hybridomas herein termed 10D1,4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, 7A4, and 7A4D. Thus, thepresent invention also encompasses the hybridomas 10D1, 4C10, 7D3, 13F1,15H5, 14A6, 22A5, 6E1, 5A8, 7A4, and 7A4D, as well as any hybridomaproducing an antibody disclosed herein. The present invention alsoprovides isolated polynucleotides encoding the antibodies and fragmentsthereof provided herein. Expression vectors comprising the isolatedpolynucleotides, and host cells comprising such expression vectors, arealso encompassed in the invention.

In one embodiment, the present invention provides anti-PD-1 antibodyimmunoconjugates. Thus, the present invention provides an antibody orfragment thereof that binds to PD-1 and that is linked or conjugated toa therapeutic agent. Therapeutic agents that may be linked or conjugatedto the anti-PD-1 antibody may include, but are not limited to, cytotoxicdrugs, radioactive isotopes, immunomodulators, or antibodies.

In one aspect, the present invention provides compositions comprisingone or more anti-PD-1 antibody or fragment thereof provided herein, anda pharmaceutically acceptable carrier.

In one aspect, the present invention provides methods for modulating animmune response in a subject, the method comprising administering to thesubject a therapeutically effective amount of an anti-PD-1 antibody orfragment thereof provided herein. In one embodiment, the presentinvention provides methods for treating or preventing a disease ordisorder in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of an anti-PD-1 antibody orfragment thereof provided herein.

In one embodiment, the present invention provides a method for enhancinganti-tumor responses in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ananti-PD-1 antibody or fragment of the invention. In another embodiment,the present invention provides a method for reducing tumors orinhibiting the growth of tumor cells in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of an anti-PD-1 antibody or fragment of the invention. In anotherembodiment, the present invention provides a method for treating cancerin a subject in need thereof, comprising administering to the subject atherapeutically effective amount of an anti-PD-1 antibody or fragment ofthe invention. In a further embodiment, the cancer is selected from thegroup consisting of lymphoma, leukemia, melanoma, glioma, breast cancer,lung cancer, colon cancer, bone cancer, ovarian cancer, bladder cancer,kidney cancer, liver cancer, stomach cancer, rectal cancer, testicularcancer, salivary cancer, thyroid cancer, thymic cancer, epithelialcancer, head or neck cancer, gastric cancer, pancreatic cancer, or acombination thereof.

In one embodiment, the present invention provides a method for treatingan infectious disease in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ananti-PD-1 antibody or fragment of the invention. In a furtherembodiment, the infectious disease is selected from the group consistingof candidiasis, candidemia, aspergillosis, streptococcal pneumonia,streptococcal skin and oropharyngeal conditions, gram negative sepsis,tuberculosis, mononucleosis, influenza, respiratory illness caused byRespiratory Syncytial Virus, malaria, schistosomiasis, andtrypanosomiasis.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are a graphs showing the blockage of PD-1 ligand PD-L1and PD-L2 binding to PD-1 by murine anti-PD-1 antibodies as measured byFACS. FIG. 1A shows the blockage of PD-L1's binding by murine anti-PD-1antibodies and FIG. 1B shows the blockage of PD-L2's binding by murineanti-PD-1 antibodies. The top panels of FIG. 1A and FIG. 1B show the MFIover a range of antibody concentrations. The blockage IC50 for theanti-PD-1 antibodies are shown in the bottom panels of FIG. 1A and FIG.1B.

FIG. 2 is a graph showing IL-2 (pg/mL) production in an MLR in responseto different concentrations of murine anti-PD-1 antibodies. Theanti-PD-1 antibodies tested were, from left to right, control mIgG1,22A5-mIgG1, 6E-mIgG1, 10D1-mIgG1, 4C10-mIgG1, 7D3-mIgG1, 13F1-mIgG1,14A6-mIgG1, 15H5-mIgG1, 5A8-mIgG1, and 7A4-mIgG1. As shown on thex-axis, each antibody was tested at 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02μg/mL, and 0.002 μg/mL.

FIG. 3 is a graph showing IFN-γ (pg/mL) production in an MLR in responseto different concentrations of murine anti-PD-1 antibodies. Theanti-PD-1 antibodies tested were, from left to right, control mIgG1,22A5-mIgG1, 6E-mIgG1, 10D1-mIgG1, 4C10-mIgG1, 7D3-mIgG1, 13F1-mIgG1,14A6-mIgG1, 15H5-mIgG1, 5A8-mIgG1, and 7A4-mIgG1. As shown on thex-axis, each antibody was tested at 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02μg/mL, and 0.002 μg/mL.

FIG. 4 is a graph showing IL-2 (pg/mL) production in an MLR in responseto different concentrations of chimeric anti-PD-1 antibodies. Thechimeric anti-PD-1 antibodies tested were, from left to right, controlhIgG4, chimeric 4C10-hIgG4, chimeric 6E1-hIgG4, chimeric 7A4-hIgG4,chimeric 13F1-hIgG4, chimeric 15H5-hIgG4, chimeric 22A5-hIgG4, andchimeric 7D3-hIgG4. As shown on the x-axis, each antibody was tested at20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL, and 0.002 μg/mL.

FIG. 5 is a graph showing IFN-γ (pg/mL) production in an MLR in responseto different concentrations of chimeric anti-PD-1 antibodies. Thechimeric anti-PD-1 antibodies tested were, from left to right, controlhIgG4, chimeric 4C10-hIgG4, chimeric 6E-hIgG4, chimeric 7A4-hIgG4,chimeric 13F1-hIgG4, chimeric 15H5-hIgG4, chimeric 22A5-hIgG4, andchimeric 7D3-hIgG4. As shown on the x-axis, each antibody was tested at20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL, and 0.002 μg/mL.

FIG. 6 shows the binding EC50 of humanized 13F1 (FIG. 6A) and humanized7A4 (FIG. 6B) anti-PD-1 antibodies as measured by ELISA. The top panelof FIG. 6A shows the absorbance over a range of concentrations ofchimeric 13F1, humanized 13F1-hIgG1 (h13F1-IgG1), humanized 13F1-hIgG4(h13F1-IgG4), or control hIgG4. The bottom panel of FIG. 6A shows thecalculated EC50 of each of the test antibodies. The top panel of FIG. 6Bshows the absorbance over a range of concentrations of chimeric7A4-hIgG1, chimeric 7A4-hIgG4, humanized 784-hIgG1 (h7A4hIgG1),humanized 7A4-hIgG4 (h7A4hIgG4), or control hIgG4. The bottom panel ofFIG. 6B shows the calculated EC50 of each of the test antibodies.

FIG. 7 shows the binding EC50 of humanized 13F1 (FIG. 7A) and humanized7A4 (FIG. 7B) anti-PD-1 antibodies as measured by FACS. The top panel ofFIG. 7A shows the mean fluorescence intensity (MFI) over a range ofconcentrations of control hIgG4, chimeric 13F1-hIgG4, humanized13F1-hIgG1 (h13F1-hIgG1), or humanized 13F1-hIgG4 (h13F1-hIgG4). Thebottom panel of FIG. 7A shows the calculated EC50 of each of the testantibodies. The top panel of FIG. 7B shows the MFI over a range ofconcentrations of control hIgG4, chimeric 7A4-hIgG4, chimeric7A4-chimeric-IgG1, humanized 7A4-IgG4 (h7A4-hIgG4), or humanized7A4-IgG1 (h7A4-hIgG1). The bottom panel of FIG. 7B shows the calculatedEC50 of each of the test antibodies.

FIG. 8 shows the blockage of PD-L1 binding by humanized 13F1 (FIG. 8A)and humanized 7A4 (FIG. 8B) anti-PD-1 antibodies as measured by ELISA.FIG. 8A shows the absorbance over a range of concentrations of controlhIgG4, chimeric 13F1, humanized 13F1-hIgG1, or humanized 13F1-hIgG4.FIG. 8B shows the absorbance over a range of concentrations of controlhIgG4, chimeric 7A4-hIgG1, chimeric 7A4-hIgG4, humanized 784-hIgG1 orhumanized 7A4-hIgG4. FIG. 8C shows the calculated PD-L1 blockage IC50 ofthe chimeric and humanized 13F1 and 7A4 antibodies.

FIG. 9 shows the blockage of PD-L1 binding by humanized 13F1 and 7A4antibodies as measured by FACS. The top panel of FIG. 9 shows the MFIover a range of antibody concentrations. The blockage IC50 for thehumanized antibodies are shown in the bottom panel of FIG. 9 .

FIG. 10 shows the binding data for PD-1 humanized monoclonal antibodiesh13F1 (top left panel) and h7A4 (top right panel), as measured byBiacore assay. The bottom panel provides the quantified binding data asmeasured by Biacore assay.

FIG. 11 is a graph showing IL-2 production (pg/mL) in an MLR reaction inthe presence of control hIgG4, murine 13F1-mIgG1 (13F1-mIgG1), humanized13F1-hIgG1, humanized 13F1-hIgG4, or chimeric 7A4-hIgG4 at the followingconcentrations: 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL, and 0.002μg/mL.

FIG. 12 is a graph showing IFN-γ production (pg/mL) in an MLR reactionin the presence of control hIgG4, murine 13F1-mIgG1(13F1-mIgG1),humanized 13F1-hIgG1, humanized 13F1-hIgG4, or chimeric 7A4-hIgG4 at thefollowing concentrations: 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL, and0.002 μg/mL.

FIG. 13 is a graph showing IL-2 production (pg/mL) in an MLR reaction inthe presence of control hIgG4, chimeric 7A4-hIgG1, chimeric 7A4-hIgG4,humanized 7A4-hIgG1, or humanized 7A4-hIgG4 at the followingconcentrations: 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL, and 0.002μg/mL.

FIG. 14 is a graph showing IFN-γ production (pg/mL) in an MLR reactionin the presence of control hIgG4, chimeric 7A4-hIgG1, chimeric7A4-hIgG4, humanized 7A4-hIgG1, or humanized 7A4-hIgG4 at the followingconcentrations: 20 μg/mL, 2 μg/mL, 0.2 μg/mL, 0.02 μg/mL, and 0.002μg/mL.

FIG. 15 shows the effect of humanized anti-PD-1 antibodies on memory Tcell responses recalled by tetanus toxin, as measured by IFN-γproduction (pg/mL). Negative control hIgG4, humanized 13F1-hIgG1,humanized 13F1-hIgG4, humanized 7A4-hIgG1, and humanized 7A4-hIgG4antibodies were tested at the following concentrations: 20 μg/mL, 2μg/mL, 0.2 μg/mL, 0.02 μg/mL, and 0.002 μg/mL.

FIG. 16 shows IFN-γ production (pg/mL) from T cells in response tocostimulation with autologous DCs and anti-CD3 antibody, in the presenceof 10 μg/ml of humanized anti-PD-1 antibodies (h13F1-hIgG1, h13F1-hIgG4,h7A4-hIgG1, or h7A4-hIgG4), isotype control (hIgG4) antibody, or noantibody.

FIGS. 17A and 17B show the data of Biacore based binding (FIG. 17A) andFACS based blockage (FIG. 17B) for PD-1 humanized monoclonal antibodiesh7A4 and h7A4D. For FIG. 17A, top left indicates h7A4 and top rightindicates 7A4D, and the bottom panel of FIG. 17A provides the quantifiedbinding data as measured by Biacore analysis. FIG. 17B indicates theblockage IC50 of PD-L1's binding to 293T-PD1 cells by 7A4D-hIgG4antibody.

FIG. 18 is a graph showing IL-2 production (pg/mL) in an MLR reaction inthe presence of control hIgG4, humanized 7A4-hIgG4, or humanized7A4D-hIgG4 at the following concentrations: 20 μg/mL, 2 μg/mL, 0.2μg/mL, 0.02 μg/mL, and 0.002 μg/mL.

FIG. 19 is a graph showing IFN-γ production (pg/mL) in an MLR reactionin the presence of control hIgG4, humanized 7A4-hIgG4, or humanized7A4D-hIgG4 at the following concentrations: 20 μg/mL, 2 μg/mL, 0.2μg/mL, 0.02 μg/mL, and 0.002 μg/mL.

DETAILED DESCRIPTION

Programmed death receptor 1 (PD-1) is a checkpoint receptor of immunesystem. It is primarily expressed on activated T and B cells, but alsooccurs on monocytes and CD4-CD8-double negative T cells and NK-T cellsunder thymic development (Agata et al., supra; Okazaki et al. (2002)Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol170:711-8). PD-1 has two ligands, PD-L1 and PD-L2. The interaction ofPD-1 with either of the two ligands has been shown to attenuate T-cellresponses in vitro and in vivo, which can, however, be reversed byinhibiting the local interaction of PD-1 with PD-L1, and the effect isadditive when the interaction of PD-1 with PD-L2 is blocked as well(Iwai et al. (2002) Proc. Nat'l. Acad Sci. USA 99: 12293-7; Brown et al.(2003) J. Immunol. 170:1257-66).

PD-1 has been found to have a correlation with cancer growth anddevelopment due to its role in protecting tumor cells from efficientimmune destruction. Its ligand, PD-L1, has been revealed to havesignificant expression on a number of mouse and human tumors, which ispostulated to mediate immune evasion (Iwai, Y. et al., Proc. Natl. Acad.Sci. USA. 99: 12293-12297 (2002); Strome S. E. et al., Cancer Res.,63:6501-6505 (2003); Dong et al. (2002) Nat. Med. 8:787-9). In humans,expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 (ontumor cells) has been found in a number of primary tumor biopsies asassessed by immunohistochemistry. Such tissues include cancers of thelung, liver, ovary, cervix, skin, colon, glioma, bladder, breast,kidney, esophagus, stomach, oral squamous cell, urothelial cell, andpancreas as well as tumors of the head and neck (Brown J. A. et al., J.Immunol. 170: 1257-1266 (2003); Dong H. et al., Nat. Med. 8: 793-800(2002); Wintterle et al., Cancer Res. 63:7462-7467 (2003); Strome S. E.et al., Cancer Res., 63: 6501-6505 (2003); Thompson R. H. et al., CancerRes. 66: 3381-5(2006); Thompson et al., Clin. Cancer Res. 13:1757-61(2007); Nomi T. et al., Clin. Cancer Res. 13: 2151-7. (2007)).More strikingly, PD-1 ligand expression on tumor cells has beencorrelated to poor prognosis of cancer patients across multiple tumortypes (reviewed in OkaZaki and Honjo, Int. Immunol. 19: 813-824 (2007)).

While the interaction between PD-1 and PD-L1 results in a decrease intumor infiltrating lymphocytes, a decrease in T-cell receptor mediatedproliferation, and immune evasion by the cancerous cells (Dong et al.(2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol.Immunother. 54: 3 07-3 14; Konishi et al. (2004) Clin. Cancer Res.10:5094-100), blockade of the PD-1/PD-L1 interaction was accordinglyshown to enhance tumor-specific T-cell immunity and be helpful inclearance of tumor cells by the immune system. In a murine model ofaggressive pancreatic cancer, for example, Nomi T., et al. (Clin. CancerRes. 13: 2151-2157, 2007) demonstrated the therapeutic efficacy ofPD-1/PD-L1 blockade. Administration of either PD-1 or PD-L1 directedantibody significantly inhibited tumor growth. Antibody blockadeeffectively promoted tumor reactive CD8+ T cell infiltration into thetumor resulting in the up-regulation of anti-tumor effectors includingIFN-γ, granzyme B and perforin. Additionally, the authors showed thatPD-1 blockade can be effectively combined with chemotherapy to yield asynergistic effect. In another study, using a model of squamous cellcarcinoma in mice, antibody blockade of PD-1 or PD-L1 significantlyinhibited tumor growth (Tsushima F. et al., Oral Oncol. 42:268-274(2006)).

Furthermore, transfection of a murine mastocytoma line with PD-L1 led todecreased lysis of the tumor cells when co-cultured with atumor-specific CTL clone. Lysis was restored when anti-PD-L1 mAb wasadded (Iwai Y. et al., Proc. Natl. Acad. Sci. USA. 99: 12293-12297(2002)). In vivo, blocking the PD1/PD-L1 interaction was shown toincrease the efficacy of adoptive T cell transfer therapy in a mousetumor model (Strome S. E. et al., Cancer Res. 63:6501-6505 (2003)).Further evidence for the role of PD-1 in cancer treatment comes fromexperiments performed with PD-1 knockout mice. PD-L1 expressing myelomacells grew only in Wild-type animals (resulting in tumor growth andassociated animal death), but not in PD-1 deficient mice (Iwai Y., etal., Proc. Natl. Acad. Sci. USA. 99: 12293-12297(2002)). In humanstudies, R. M. Wong et al. (Int. Immunol. 19:1223-1234 (2007)) showedthat PD-1 blockade using a fully human anti-PD-1 antibody augmented theabsolute numbers of tumor-specific CD8+ T cells (CTLs) in ex vivostimulation assays using vaccine antigens and cells from vaccinatedindividuals. In a similar study, antibody blockade of PD-L1 resulted inenhanced cytolytic activity of tumor-associated antigen-specificcytotoxic T cells and increased cytokine production by tumor specific THcells (Blank C. et al., Int. J. Cancer 119: 317-327 (2006)). The sameauthors showed that PD-L1 blockade augments tumor-specific T cellresponses in vitro when used in combination with anti-CTLA-4 blockade.Overall, the PD-1/PD-L1 pathway is a target for the development ofantibody therapeutics for cancer treatment. Anti-PD-1 antibodies mayalso be useful in chronic viral infection. Memory CD8+ T cells generatedafter an acute viral infection are highly functional and constitute animportant component of protective immunity. In contrast, chronicinfections are often characterized by varying degrees of functionalimpairment (exhaustion) of virus-specific T-cell responses, and thisdefect is a principal reason for the inability of the host to eliminatethe persisting pathogen. Although functional effector T cells areinitially generated during the early stages of infection, they graduallylose function during the course of a chronic infection. Barber et al.(Barber et al., Nature 439: 682-687 (2006)) showed that mice infectedwith a laboratory strain of LCMV developed chronic infection resultingin high levels of virus in the blood and other tissues. These miceinitially developed a robust T cell response, but eventually succumbedto the infection upon T cell exhaustion. The authors found that thedecline in number and function of the effector T cells in chronicallyinfected mice could be reversed by injecting an antibody that blockedthe interaction between PD-1 and PD-L1.

In one aspect, the present invention provides antibodies or antigenbinding fragments thereof that bind to programmed cell death 1 (PD-1).PD-1. In one embodiment, the antibodies or fragments thereof bind tohuman PD-1. In another embodiment, the antibodies or fragments thereofbind to human and to cynomolgous PD-1. In another embodiment, theantibodies or fragments thereof block the interaction of PD-1 on T cellswith its ligand PD-L1. In one aspect, the present invention providesmethods of making and using the anti-PD-1 antibodies or fragmentsthereof, and compositions comprising anti-PD-1 antibodies or fragmentsthereof, including pharmaceutical compositions.

As used herein, the term “antibody” refers to a binding protein havingat least one antigen binding domain. The antibodies and fragmentsthereof of the present invention may be whole antibodies or any fragmentthereof. Thus, the antibodies and fragments of the invention includemonoclonal antibodies or fragments thereof and antibody variants orfragments thereof, as well as immunoconjugates. Examples of antibodyfragments include Fab fragments, Fab′ fragments, F(ab)′ fragments, Fvfragments, isolated CDR regions, single chain Fv molecules (scFv), andother antibody fragments known in the art. Antibodies and fragmentsthereof may also include recombinant polypeptides, fusion proteins, andbi-specific antibodies. The anti-PD-1 antibodies and fragments thereofdisclosed herein may be of an IgG1, IgG2, IgG3, or IgG4 isotype. Theterm “isotype” refers to the antibody class encoded by the heavy chainconstant region genes. In one embodiment, the anti-PD-1 antibodies andfragments thereof disclosed herein are of an IgG1 or an IgG4 isotype.The PD-1 antibodies and fragments thereof of the present invention maybe derived from any species including, but not limited to, mouse, rat,rabbit, primate, llama, and human. The PD-1 antibodies and fragmentsthereof may be chimeric, humanized, or fully human antibodies. In oneembodiment, the anti-PD-1 antibodies are murine antibodies. In anotherembodiment, the anti-PD1 antibodies are chimeric antibodies. In afurther embodiment, the chimeric antibodies are mouse-human chimericantibodies. In another embodiment, the antibodies are derived from miceand are humanized.

A “chimeric antibody” is an antibody having at least a portion of theheavy chain variable region and at least a portion of the light chainvariable region derived from one species; and at least a portion of aconstant region derived from another species. For example, in oneembodiment, a chimeric antibody may comprise murine variable regions anda human constant region.

A “humanized antibody” is an antibody containing complementaritydetermining regions (CDRs) that are derived from a non-human antibody;and framework regions as well as constant regions that are derived froma human antibody. For example, the anti-PD-1 antibodies provided hereinmay comprise CDRs derived from one or more murine antibodies and humanframework and constant regions. Thus, in one embodiment, the humanizedantibody provided herein binds to the same epitope on PD-1 as the murineantibody from which the antibody's CDRs are derived. Exemplary humanizedantibodies are provided herein. Additional anti-PD-1 antibodiescomprising the heavy and light chain CDRs provided herein, or variantsthereof, may be generated using any human framework sequence, and arealso encompassed in the present invention. In one embodiment, frameworksequences suitable for use in the present invention include thoseframework sequences that are structurally similar to the frameworksequences provided herein. Further modifications in the frameworkregions may be made to improve the properties of the antibodies providedherein. Such further framework modifications may include chemicalmodifications; point mutations to reduce immunogenicity or remove T cellepitopes; or back mutation to the residue in the original germlinesequence.

In some embodiments, such framework modifications include thosecorresponding to the mutations exemplified herein, includingbackmutations to the germline sequence. For example, in one embodiment,one or more amino acids in the human framework regions of the VH and/orVL of the humanized antibodies provided herein are back mutated to thecorresponding amino acid in the parent murine antibody. As an example,as for VH and VL of 7A4 and 13F1, several sites of framework amino acidof the aforementioned template human antibody were back mutated to thecorresponding amino acid sequences in mouse 7A4 and 13F1 antibody. Inone embodiment, the amino acid at positions 40 and/or 45 and/or 70and/or 72 of the light chain variable region is back mutated to thecorresponding amino acid found at that position in the mouse 7A4 or 13F1light chain variable region. In another embodiment, the amino acid atpositions 2 and/or 26 and/or 46 and/or 48 and/or 49 and/or 67 and/or 70and/or 71 of the heavy chain variable region is back mutated to thecorresponding amino acid found at that position in the mouse 7A4 or 13F1heavy chain variable region. In one embodiment, the humanized 7A4antibody comprises a light chain variable region wherein the amino acidat position 40 is mutated from Tyr (Y) to Phe (F) and the amino acid atposition 72 is mutated from Gly (G) to Arg (R); and a heavy chainvariable region wherein the amino acid at position 2 is mutated from Val(V) to Ile (I), the amino acid at position 46 is mutated from Glu (E) toLys (K), and the amino acid at position 70 is mutated from Phe (F) toIle (I). In one embodiment, the humanized 13F1 antibody comprises alight chain variable region wherein the amino acid at position 45 ismutated from Leu (L) to Pro (P) and the amino acid at position 70 ismutated from Phe (F) to Tyr (Y); and a heavy chain variable regionwherein the amino acid at position 26 is mutated from Gly (G) to Tyr(Y), the amino acid at position 48 is mutated from Ile (I) to Met (M),the amino acid at position 49 is mutated from Gly (G) to Ala (A), theamino acid at position 67 is mutated from Val (V) to Ile (I), and theamino acid at position 71 is mutated from Val (V) to Arg (R). Additionalor alternate back mutations may be made in the framework regions of thehumanized antibodies provided herein in order to improve the propertiesof the antibodies.

The present invention also encompasses humanized antibodies that bind toPD-1 and comprise framework modifications corresponding to the exemplarymodifications described herein with respect to any suitable frameworksequence, as well as other framework modifications that otherwiseimprove the properties of the antibodies. For example, in someembodiments, the antibodies provided herein comprise one or moremutations to remove one or more deamidation sites or one or moreoxidation sites. For example, in one embodiment, the antibodies providedherein comprise a mutation of one or more asparagine residues to removeone or more deamidation sites; and/or mutation of one or more methionineresidues to remove one or more oxidation sites.

In other embodiments, the antibodies provided herein comprise one ormore mutations to improve stability, improve solubility, alterglycosylation, and/or reduce immunogenicity, such as, for example, bytargeted amino acid changes that reduce deamidation or oxidation, reduceisomerization, optimize the hydrophobic core and/or charge clusterresidues, remove hydrophobic surface residues, optimize residuesinvolved in the interface between the variable heavy and variable lightchains, and/or modify the isoelectric point.

As used herein, the term “derived” when used to refer to a molecule orpolypeptide relative to a reference antibody or other binding protein,means a molecule or polypeptide that is capable of binding withspecificity to the same epitope as the reference antibody or otherbinding protein.

The antibodies and antigen-binding fragments thereof disclosed hereinare specific for PD-1. In one embodiment, the antibodies and fragmentsthereof are specific for human PD-1. In one embodiment, the antibodiesand fragments provided herein bind to human or primate PD-1 but not toPD-1 from any other mammal. In a further embodiment, the antibodies andfragments thereof do not bind to mouse PD-1. The terms “human PD-1,”“hPD-1”, and “huPD-1” and the like are used interchangeably herein andrefer to human PD-1 and variants or isoforms of human PD-1. By “specificfor” is meant that the antibodies and fragments thereof bind PD-1receptor with greater affinity than any other target. In one embodiment,the PD-1 antibodies and fragments provided herein are specific for PD-1and do not cross react with CTLA4, ICOS, or CD28. As used herein, theterm “EC50” refers to the effective concentration, 50% maximal responseof the antibody. As used herein, the term “IC50” refers to theinhibitory concentration, 50% maximal response of the antibody. BothEC50 and IC50 may be measured by ELISA or FACS analysis, or any othermethod known in the art.

In one embodiment, the anti-PD1 antibodies and fragments or variantsthereof have an affinity (KD) for PD-1 in the range of about 0.001 nM toabout 100 nM, about 0.002 nM to about 50 nM, about 0.005 nM to about 5nM, about 0.01 nM to about 1 nM, or about 0.05 nM to about 0.1 nM. Inone embodiment, the antibodies and fragments thereof have an affinity(KD) for PD-1 of about 50 nM or less, about 25 nM or less, about 20 nMor less, about 15 nM or less, about 10 nM or less, about 8 nM or less,about 6 nM or less, about 4 nM or less, about 2 nM or less, about 1 nMor less, about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM orless, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less,about 0.3 nM or less, about 0.2 nM or less, about 0.1 nM or less, about0.09 nM or less, about 0.08 nM or less, about 0.07 nM or less, about0.06 nM or less, about 0.05 nM or less, about 0.04 nM or less, about0.03 nM or less, about 0.02 nM or less, about 0.01 nM or less, about0.009 nM or less, about 0.008 nM or less, about 0.007 nM or less, about0.006 nM or less, about 0.005 nM or less, about 0.004 nM or less, about0.003 nM or less, about 0.002 nM or less, or about 0.001 nM or less. Inone embodiment, the antibodies and fragments thereof have an affinity(KD) for PD-1 of about 10 nM, about 9 nM, about 8 nM, about 7 nM, about6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.09 nM, about0.08 nM, about 0.07 nM, about 0.06 nM, about 0.05 nM, about 0.04 nM,about 0.03 nM, about 0.02 nM, about 0.01 nM, about 0.009 nM, about 0.008nM, about 0.007 nM, about 0.006 nM, about 0.005 nM, about 0.004 nM,about 0.003 nM, about 0.002 nM, or about 0.001.

In one embodiment, the antibodies and fragments provided herein comprisealight chain and a heavy chain, each of which comprises three CDRregions. Exemplary light chain CDR sequences (LCDR1, LCDR2, and LCDR3)for PD-1 antibodies of the invention are provided below in Table 1.Exemplary heavy chain CDR sequences (HCDR1, HCDR2, and HCDR3) for PD-1antibodies of the invention are provided below in Table 2. Exemplaryvariable regions and full antibody sequences for PD-1 antibodies of theinvention are provided below in Table 3.

TABLE 1 Light Chain CDR sequences Name LCDR SEQ ID NO Sequence 10D1 1 24 RASQSISNNLH 2  25 YASQSIS 3  26 QQSNSWPLT 4C10 1  34 KASQSVSDDVA 2 35 YAFNRYT 3  36 QQDYRSPWT 7D3 1  45 RASQSISNDLH 2  46 YVSQSIS 3  47QQSDSWPLT 13F1 1  55 RANSSVSSMH 2  56 AISNLAF 3  57 QQWSSRPPT 15H5 1  65HASQSINVWLS 2  66 ASNLHT 3  67 QQGQSYPWT 14A6 1  75 RANSSVSSMH 2  76AISNLAF 3  77 QQWNSRPPT 22A5 1  85 KASQDVDNAVA 2  86 WASTRHH 3  87QQYSTFPYT 6E1 1  95 RASQSLSNNLH 2  96 YASQSIS 3  97 QQSNSWPLT 5A8 1 105KASQSVSNDVA 2 106 YAFTRYI 3 107 QQDYSSPYT 74A 1 115 RASWSVDNYGYSFMN 2116 RASNLES 3 117 QQSNADPT

TABLE 2 Heavy chain CDR sequences Name HCDR SEQ ID NO Sequence 10D1 1 19SYGMS 2 20 TMSGGGRDIYYPDSMKG 3 21 QYYDDWFAY 4C10 1 29 TYGVH 2 30VIWSGGSTDYNAAFIS 3 31 EKSVYGNYVGAMDY 7D3 1 40 SYGMS 2 41TISGGGRDIYYPDSVKG 3 42 QYYDDWFAY 13F1 1 50 SDYAWN 2 51 YISYSGYTSYNPSLKS3 52 SLDYDYGTMDY 15H5 1 60 SYDMS 2 61 TISGGGSYTYYQDSVKG 3 62 PYGPYFDY14A6 1 70 SDYAWN 2 71 YISYSGYTSYNPSLKS 3 72 SLDYDYGTMDY 22A5 1 80 YYDMS2 81 TISGGGRNTYFIDSVKG 3 82 PYEGAVDF 6E1 1 90 SYGMS 2 91TISGGGRDTYYLDSVKG 3 92 QYYDDWFAY 5A8 1 100 NNWIG 2 101 DFYPGGGYTNYNEKFKG3 102 GYGTNYWYFDV 7A4 1 110 NFGMN 2 111 WISGYTREPTYAADFKG 3 112 DVFDY

TABLE 3 Light chain and heavy chain variable regionsequences and full antibody sequences Name Region¹ SEQ ID NO Sequence10D1 Light 23 D I V L T Q T P A T L S V T P G D S V S L S C R A murinechain S Q S I S N N L H W Y Q Q K S H E S P R L L I K Y variableA S Q S I S G I P S R F S G S G S G T D F T L N IN S V E T E D F G M Y F C Q Q S N S W P L T F G A G T K L E L K R 10D1Heavy 18 E V K L V E S G G G L V K P G G S L K L S C A A S murine chainG F T F S S Y G M S W L R Q T P E K R L E W V A T variableM S G G G R D I Y Y P D S M K G R F T I S R D N AK N N L Y L Q M S S L R S E D T A L Y Y C A R Q YY D D W F A Y W G Q G T L V T V S A 4C10 Light 33S I V M T Q T P K F L L V S A G D R V T I T C K A murine chainS Q S V S D D V A W Y Q Q K P G Q S P K L L I Y Y variableA F N R Y T G V P D R F T G S G Y G T D F T F T IS T V Q S E D L A V Y F C Q Q D Y R S P W T F G G G T K L E I K R 4C10Heavy 28 Q V Q L K Q S G P G L V Q P S Q N L S V T C T V S murine chainG F S L T T Y G V H W V R Q S P G K G L E W L G V variableI W S G G S T D Y N A A F I S R L T I S K D N A RS Q V F F K M N S L Q V N D T A M Y Y C A R E K SV Y G N Y V G A M D Y W G Q G T S V T V S S 7D3 Light 44D I V L T Q S P A T L S V T P G D S V S L S C R A murine chainS Q S I S N D L H W Y Q Q K S H E S P R L L I K Y variableV S Q S I S G I P S R F S G S G S G T D F T L S IN S V E T E D F G M Y F C Q Q S D S W P L T F G A G T K L E L K R 7D3Heavy 39 E V K L V E S G G G L V K P G G S L K L S C G A S murine chainG F T F S S Y G M S W V R Q T P E K R L E W V A T variableI S G G G R D I Y Y P D S V K G R L T I S R D N AK N N L Y L Q M S S L R S E D T A L Y Y C V R Q yY D D W F A Y W G Q G T L V T V S A 13F1 Light 54Q I V L S Q S P A I L S A S P G E K V T M T C R A murine chainN S S V S S M H W Y Q Q K P G S S P E P W I Y A I variableS N L A F G V P T R F S G S G S G T S Y S L T I SR V E A E D A A T Y F C Q Q W S S R P P T F G G G T K L E I K R 13F1Heavy 49 D V Q L Q E S G P G L V K P S Q S L S L T C T V T murine chainG Y S I T S D Y A W N W I R Q F P G N Q L E W M A variableY I S Y S G Y T S Y N P S L K S R I S I T R D T SK N Q F F L Q L N S V T T E D T A T Y Y C A R S LD Y D Y G T M D Y W G Q G T S V T V S S 15H5 Light 64D I Q M N Q S P S S L S A S L G D T I T I T C H A  murine chainS Q S I N V W L S W Y Q Q K P G N I P K L L I Y R variableA S N L H T G V P S R F S G S G S G T G F T L T IS S L Q P D D I A T Y Y C Q Q G Q S Y P W T F G G G T K L E I K R 15H5Heavy 59 E V K L V E S G G G L V K P G G S L K L S C A A S murine chainG F A F R S Y D M S W V R Q T P E K I L E W V A T variableI S G G G S Y T Y Y Q D S V K G R F T I S R D N AR N T L Y L Q M S S L R S E D T A L Y Y C A S P Y G P Y F D Y W G Q G T T L T V S S 14A6 Lgight 74Q I V L S Q S P A I L S A S P G E K V T M T C R A murine chainN S S V S S M H W Y Q Q K P G S S P E P W I Y A I variableS N L A F G V P A R F S G S G S G T S Y S L T I SR V E A E D A A T Y F C Q Q W N S R P P T F G G G T K L E I K R 14A6Heavy 69 D V Q L Q E S G P G L V K P S Q S L S L T C T V T murine chainG Y S I T S D Y A W N W I R Q F P G N Q L E W M A variableY I S Y S G Y T S Y N P S L K S R I S I T R D T SR N Q F F L Q L N S V T T E D T A T Y Y C A R S LD Y D Y G T M D Y W G Q G T S V T V S S 22A5 Ligght 84D I V M T Q S H K V M S T S V G D R V S I T C K A murine chainS Q D V D N A V A W Y Q Q N P G Q S P K L L I K WA S T R H H G V P D R F T G S G S G T D F T L T IS T V Q S E D L A D F F C Q Q Y S T F P Y T F G G  G T K L E I K R 22A5Heavy 79 E V K L V E S G G G L V K P G G S L K L S C S A S murine chainG F S F S Y Y D M S W V R Q T P E K G L E W V A T variableI S G G G R N T Y F I D S V K G R F T I S R D N VK N N L Y L L M S S L R S E D T A L Y Y C A S P Y E G A V D F W G Q G T S V T V S S 6E1 Light 94D I V L T Q T P A T L S V T P G D S V S L S C R A murine chainS Q S L S N N L H W Y Q Q K S H E S P R L L I K Y variableA S Q S I S G I P S R F S G S G S G T D F T L S IN S V E T E D F G M Y F C Q Q S N S W P L T F G A G T K L E M K R 6E1Heavy 89 E V K L V E S G G G L V K P G G S L K L S C A A S murine chainG F T F S S Y G M S W V R Q T P E K R L E W V A T variableI S G G G R D T Y Y L D S V K G R F T I S R D N AK N N L Y L Q M S S L R S E D T A L Y Y C V R Q YY D D W F A Y W G Q G T L V S N S A 5A8 Light 104N I V M T Q T P K I L F I S A G D R V T I T C K A murine chainS Q S V S N D V A W Y Q Q K P G Q S P K L L I Y Y variableA F T R Y I G V P D R F T G S G Y G T D F T F T IS T V Q A E D L A V Y F C Q Q D Y S S P Y T F G G G T K L E I K R 5A8Heavy 99 Q V Q L Q Q S G D E L V R P G T S V K M S C K A A murine chainG Y T F T N N W I G W V K Q R P G H G L E W I G D variableF Y P G G G Y T N Y N E K F K G K A T L T A D T SS S T A Y M Q L S S L T S E D S A I Y Y C A R G YG T N Y W Y F D V W G A G T T V T V S S 7A4 Light 114D I V L T Q S P A S L A V S L G Q R A T I S C R A murine chainS E S V D N Y G Y S F M N W F Q Q K P G Q P P K L variableL I Y R A S N L E S G I P A R F S G S G S R T N FT L T I N P V E A D D V A T Y F C Q Q S N A D P TF G G G T N L E I K R A 7A4 Heavy 109Q I H L V Q S G P E L K K P G E T V K I S C K A S murine chainG Y T F T N F G M N W V K Q A P G K G L K W M G W variableI S G Y T R E P T Y A A D F K G R F A I S L E T SA S T A Y L Q I N D L K N E D M A T Y F C A R D V F D Y W G Q G T T L T V S S 7A4 Full 119QIHLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGW chimeric lengthISGYTREPTYAADFKGRFAISLETSASTAYLQINDLKNEDMATYFCARDV heavyFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP chainVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN IgG1HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 7A4 Full 121QIHLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGW chimeric lengthISGYTREPTYAADFKGRFAISLETSASTAYLQINDLKNEDMATYFCARDV heavyFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP chainVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD IgG4HKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 7A4 Full 123DIVLTQSPASLAVSLGQRATISCRASESVDNYGYSFMNWFQQKPGQPPKL chimeric lengthLIYRASNLESGIPARESGSGSRTNFTLTINPVEADDVATYFCQQSNADPT lightFGGGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ chainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC13F1 Full 125 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNQLEWMAchimeric length YISYSGYTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARSL heavyDYDYGTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT IgG1YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 13F1 Full 127DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNQLEWMA chimeric lengthYISYSGYTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARSL heavyDYDYGTMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK chainDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT IgG4YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 13F1 Full 129QIVLSQSPAILSASPGEKVTMTCRANSSVSSMHWYQQKPGSSPEPWIYAI chimeric lengthSNLAFGVPTRFSGSGSGTSYSLTISRVEAEDAATYFCQQWSSRPPTFGGG lightFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD chainNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 7A4Heavy 131 QIQLVQSGSELKKPGASVKVSCKASGYTFTNEGMNWVRQAPGQGLKWMGW humanizedchain ISGYTREPTYAADFKGREVISLDTSVSTAYLQISSLKAEDTAVYYCARDV variableFDYWGQGTLVTVSS 7A4 Light 133DIVLTQSPASLAVSPGQRATITCRASESVDNYGYSFMNWFQQKPGQPPKL humanized chainLIYRASNLESGVPARFSGSGSRTDFTLTINPVEANDTANYYCQQSNADPT variable FGQGTKLEIK13F1 Heavy 141 QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDYAWNWIRQPPGKGLEWMAhumanized chain YISYSGYTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARSLvariable DYDYGTMDYWGQGTLVTVSS 13F1 Light 143EIVLTQSPATLSLSPGERATLSCRANSSVSSMHWYQQKPGQSPEPWIYAI humanized chainSNLAFGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSRPPTFGQG variable TKLEIK 7A4Full 135 QIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLKWMGW humanizedheavy ISGYTREPTYAADFKGREVISLDTSVSTAYLQISSLKAEDTAVYYCARDV -IgG1 chainFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP (D265A)VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 7A4 Full 137QIQLVQSGSELKKPGASVKVSCKASGYTFTNFGMNWVRQAPGQGLKWMGW humanized heavyISGYTREPTYAADFKGREVISLDTSVSTAYLQISSLKAEDTAVYYCARDV -IgG4 chainFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP (F234A/L235A)VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 7A4 Full 139DIVLTQSPASLAVSPGQRATITCRASESVDNYGYSFMNWFQQKPGQPPKL humanized lightLIYRASNLESGVPARFSGSGSRTDFTLTINPVEANDTANYYCQQSNADPT chainFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC13F1 Full 145 QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDYAWNWIRQPPGKGLEWMAhumanized heavy YISYSGYTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARSL -IgG1chain DYDYGTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK (D265A)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 13F1 Full 147QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDYAWNWIRQPPGKGLEWMA humanized heavyYISYSGYTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARSL -IgG4 chainDYDYGTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK (F234A/L235A)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 13F1 Full 149EIVLTQSPATLSLSPGERATLSCRANSSVSSMHWYQQKPGQSPEPWIYAI humanized lightSNLAFGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSRPPTFGQG chainTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC HumanD265A 150 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV IgG1mutation HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP constantKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVS regionHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human F234A 151ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV IgG4 andHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES constant L235AKYGPPCPPCPAPEAAGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQED region doublePEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK mutationCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 7A4D Light 152DIVLTQSPASLAVSPGQRATITCRASESVDNYGYSFMNWFQQKPGQPPKL humanized chainLIYRASNLESGVPARFSGSGSRTDFTLTINPVEADDTANYYCQQSNADPT variable FGQGTKLEIK7A4D Full 153 DIVLTQSPASLAVSPGQRATITCRASESVDNYGYSFMNWFQQKPGQPPKLhumanized light LIYRASNLESGVPARFSGSGSRTDFTLTINPVEADDTANYYCQQSNADPT chainFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

In one embodiment, the invention provides anti-PD-1 antibodies thatcomprise the light chain CDRs and heavy chain CDRs of antibodies 10D1,4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, and/or 7A4. The person ofskill in the art will understand that the heavy and light chain CDRs ofthe antibodies provided herein may be independently selected, or mixedand matched, to form an antibody or binding fragment thereof comprisingany light chain CDR1, CDR2, and CDR3; and any heavy chain CDR1, CDR2,and CDR3 from the antibodies provided herein. Thus, the inventionprovides anti-PD-1 antibodies that comprise a light chain CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 24, 34, 45, 55, 65, 75, 85, 95, 105, and 115; a light chainCDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 25, 35, 46, 56, 66, 76, 86, 96, 106, and 116;a light chain CDR3 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 26, 36, 47, 57, 67, 77, 87, 97, 107, and117; a heavy chain CDR1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 19, 29, 40, 50, 60, 70, 80, 90, 100,and 110; a heavy chain CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 20, 30, 41, 51, 61, 71, 81, 91,101, and 111; and a heavy chain CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21, 31, 42, 52, 62,72, 82, 92, 102, and 112. In one embodiment, the present inventionprovides anti-PD-1 antibodies comprising heavy and light chain CDRregions comprising amino acid sequences having at least 75%, at least80%, at least at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%homology to the corresponding light or heavy chain CDR1, CDR2, or CDR3provided herein. In one embodiment, the present invention providesanti-PD-1 antibodies comprising heavy and light chain CDR regionscomprising amino acid sequences having 1, 2, 3, 4, 5, or 6 amino acidsubstitutions, deletions, or insertions relative to the correspondinglight or heavy chain CDR1, CDR2, or CDR3 provided herein.

In one embodiment, the invention provides anti-PD-1 antibodies thatcomprise a variable light chain of an antibody selected from the groupconsisting of 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, 7A4 and7A4D and a variable heavy chain of an antibody selected from the groupconsisting of 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, and7A4. In one embodiment, the antibodies and fragments provided hereincomprise a light chain variable region comprising an amino acid sequencethat is at least 75%, at least 80%, at least at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% homology to a light chain variable regionaccording to SEQ ID NOs: 23, 33, 44, 54, 64, 74, 84, 94, 104, 114, 133,143 and 152. In one embodiment, the antibodies and fragments providedherein comprise a light chain variable region comprising an amino acidsequence according to SEQ ID NO: 23, 33, 44, 54, 64, 74, 84, 94, 104,114, 133, 143, 152, or a variant thereof, wherein the variant comprises1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions,or a combination thereof. In a further embodiment, the amino acidsubstitutions are conservative substitutions. In another embodiment, theamino acid substitutions improve the properties of the antibodies asprovided herein, for example, by removing a deamidation site. Forexample, in one embodiment, an asparagine (Asn; N) residue is mutated.In a further embodiment, the Asn is mutated to aspartic acid (Asp; D).In a yet further embodiment, the Asn at position 85 in framework region3 of the light chain variable region is mutated to Asp. In oneembodiment, the present disclosure provides humanized antibody 7A4D,which comprises the same amino acid sequence as humanized antibody 7A4except with a mutation in framework 3 (position 85) of the light chainto remove the deamidation site.

In one embodiment, the antibodies and fragments provided herein comprisea heavy chain variable region comprising an amino acid sequence that isat least 75%, at least 80%, at least at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% homology to a light chain variable regionaccording to SEQ ID NOs: 18, 28, 39, 49, 59, 69, 79, 89, 99, 109, 131,and 141, or 84. In one embodiment, the antibodies and fragments providedherein comprise a heavy chain variable region comprising an amino acidsequence according to SEQ ID NO: 18, 28, 39, 49, 59, 69, 79, 89, 99,109, 131, 141, or a variant thereof, wherein the variant comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, insertions,or deletions, or a combination thereof. In a further embodiment, theamino acid substitutions are conservative substitutions. In anotherembodiment, the amino acid substitutions improve the properties of theantibodies as provided herein, for example, by removing a deamidationsite. For example, in one embodiment, an asparagine (Asn; N) residue ismutated. In a further embodiment, the Asn is mutated to aspartic acid(Asp; D).

The anti-PD-1 antibodies disclosed herein having one or more amino acidsubstitution, insertion, deletion, or combination thereof in the CDR orvariable light or heavy chain region retain the biological activity ofthe corresponding anti-PD-1 antibody that does not have an amino acidsubstitution, insertion, or deletion. Thus, the variant anti-PD-1antibodies provided herein retain binding to PD-1. Percent homology, asused herein, refers to the number of identical amino acid sequencesshared by two reference sequences, divided by the total number of aminoacid positions, multiplied by 100.

In some embodiments, the anti-PD-1 antibodies provided herein compriseconservative amino acid substitutions. The person of skill in the artwill recognize that a conservative amino acid substitution is asubstitution of one amino acid with another amino acid that has asimilar structural or chemical properties, such as, for example, asimilar side chain. Exemplary conservative substitutions are describedin the art, for example, in Watson et al., Molecular Biology of theGene, The Bengamin/Cummings Publication Company, 4^(th), Ed. (1987).

The skilled person will understand that the variable light and variableheavy chains may be independently selected, or mixed and matched, fromthe antibodies provided herein. Thus, the present invention providesanti-PD-1 antibodies comprising a light chain variable region having atleast 80% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 23, 33, 44, 54, 64, 74, 84, 94, 104, 114, 133,143, and 152; and a heavy chain variable region having at least 80%homology to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 18, 28, 39, 49, 59, 69, 79, 89, 99, 109, 131, and 141.

In one embodiment, the present invention provides antibodies that bindto the same epitope as any one of the exemplary antibodies disclosedherein. Thus, in one embodiment, the present invention providesantibodies that compete for binding to PD-1 with the exemplaryantibodies provided herein.

The anti-PD-1 antibodies and fragments thereof provided herein mayfurther comprise Fc region modifications to alter effector functions. Fcmodifications may be amino acid insertions, deletions, or substitutions,or may be chemical modifications. For example, Fc region modificationsmay be made to increase or decrease complement binding, to increase ordecrease antibody-dependent cellular cytoxicity, or to increase ordecrease the half life of the antibody. Some Fc modifications increaseor decrease the affinity of the antibody for an Fcγ receptor such asFcγRI, FcγRII, FcγRIII, or FcRn. Various Fc modifications have beendescribed in the art, for example, in Shields et al., J Biol. Chem 276;6591 (2001); Tai et al. Blood 119; 2074 (2012); Spiekermann et al. JExp. Med 196; 303 (2002); Moore et al. mAbs 2:2; 181 (2010);Medzihradsky Methods in Molecular Biology 446; 293 (2008); Mannan et al.Drug Metabolism and Disposition 35; 86 (2007); and Idusogie et al. JImmunol 164; 4178 (2000). In some embodiments, Fc region glycosylationpatters are altered. In other embodiments, the Fc region is modified bypegylation (e.g., by reacting the antibody or fragment thereof withpolyethylene glycol (PEG).

In one embodiment, the antibodies or fragments thereof provided hereinare immunoconjugates comprising an anti-PD-1 antibody or fragmentthereof and further comprising an agent selected from the groupincluding an additional therapeutic agent, a cytotoxic agent, animmunoadhesion molecule, and an imaging agent. In some embodiments, theimaging agent is selected from the group consisting of a radiolabel, anenzyme, a fluorescent label, a luminescent label, a bioluminescentlabel, a magnetic label, and biotin. In some embodiments, the imagingagent is a radiolabel selected from the group consisting of: ³H, ¹⁴C,³⁵S, ⁶², ⁶⁴Cu, ⁸⁹Zr, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and¹⁵³Sm. In some embodiments, the therapeutic agent or cytotoxic agent isselected from the group including a chemotherapeutic agent, animmunosuppressive agent, an immuno-stimulatory agent, ananti-metabolite, an alkylating agent, an antibiotic, a growth factor, acytokine, an anti-angiogenic agent, an anti-mitotic agent, ananthracycline, a toxin, and an apoptotic agent. In some embodiments, thebinding protein is conjugated directly to the agent. In otherembodiments, the binding protein is conjugated to the agent via alinker. Suitable linkers include, but are not limited to, amino acid andpolypeptide linkers disclosed herein. Linkers may be cleavable ornon-cleavable.

In one embodiment, the present invention provides bispecific ormultispecific antibodies specific for PD-1 and at least one otherantigen or epitope. The anti-PD-1 antibodies and fragments thereofprovided herein may be tested for binding to PD-1 using the bindingassays provided herein, or any other binding assay known in the art.

Unless otherwise stated, the practice of the present invention employsconventional molecular biology, cell biology, biochemistry, andimmunology techniques that are well known in the art and described, forexample, in Methods in Molecular Biology, Humana Press; MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989),Current Protocols in Immunology (J. E. Coligan et al., eds., 1991);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); Phagedisplay: a laboratory manual (C. Barbas III et al, Cold Spring HarborLaboratory Press, 2001); and Using antibodies: a laboratory manual (E.Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999).

In one aspect the present invention provides methods for treating asubject for a disease or condition responsive to enhancing, stimulating,or eliciting an immune response. As used herein, the terms “treatment”or “treating” refers to both therapeutic treatment and prophylactic orpreventive measures. Subjects in need of treatment include thosesubjects that already have the disease or condition, as well as thosethat may develop the disease or condition and in whom the object is toprevent, delay, or diminish the disease or condition. As used herein,the term “subject” denotes a mammal, such as a rodent, a feline, acanine, and a primate. Preferably, a subject according to the inventionis a human.

The term “therapeutically effective amount,” as used herein, refers tothe amount of a compound or composition that is necessary to provide atherapeutic and/or preventative benefit to the subject.

In one aspect, the antibodies and antigen binding fragments thereof areuseful in the treatment of solid or non-solid tumors. Thus, in oneaspect, the present invention provides methods for treatment of cancer.“Cancer” as used herein refers to the physiological condition in mammalsthat is typically characterized by unregulated cell growth. Examples ofcancer include but are not limited to carcinoma, lymphoma, blastoma,sarcoma (including liposarcoma, osteogenic sarcoma, angiosarcoma,endotheliosarcoma, leiomyosarcoma, chordoma, lymphangiosarcoma,lymphangioendotheliosarcoma, rhabdomyosarcoma, fibrosarcoma,myxosarcoma, chondrosarcoma), neuroendocrine tumors, mesothelioma,synovioma, schwanoma, meningioma, adenocarcinoma, melanoma, and leukemiaor lymphoid malignancies. More particular examples of such cancersinclude squamous cell cancer (e.g. epithelial squamous cell cancer),Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma, smalllymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoides,mantle cell lymphoma, follicular lymphoma, diffuse large B-celllymphoma, marginal zone lymphoma, hairy cell leukemia andlymphoplasmacytic leukemia), tumors of lymphocyte precursor cells,including B-cell acute lymphoblastic leukemia/lymphoma, and T-cell acutelymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T and NKcells, including peripheral T-cell leukemias, adult T-cellleukemia/T-cell lymphomas and large granular lymphocytic leukemia,Langerhans cell histocytosis, myeloid neoplasias such as acutemyelogenous leukemias, including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders, including chronic myelogenousleukemia, B-cell acute lymphoblastic leukemia/lymphoma, T-cell acutelymphoblastic leukemia/lymphoma, lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, small cell lung carcinoma, cancer of theperitoneum, hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophageal cancer,tumors of the biliary tract, Ewing's tumor, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, testicular tumor, lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, myelodysplastic disease, heavy chain disease,neuroendocrine tumors, Schwanoma, and other carcinomas, as well as headand neck cancer.

In one embodiment, the antibodies and fragments thereof provided hereinare useful in the treatment of diseases caused by infectious agents.Infectious agents include, but are not limited to, bacterial,mycological, parasitic, and viral agents. Examples of such infectiousagents include the following: staphylococcus, methicillin-resistantStaphylococcus aureus, Escherichia coli, streptococcaceae,neisseriaaceae, cocci, enterobacteriaceae, enterococcus,vancomycin-resistant enterococcus, cryptococcus, histoplasmosis,aspergillus, pseudomonadaceae, vibrionaceae, campylobacter,pasteurellaceae, bordetella, francisella, brucella, legionellaceae,bacteroidaceae, gram-negativebacilli, clostridium, corynebacterium,propionibacterium, gram-positive bacilli, anthrax, actinomyces,nocardia, mycobacterium, treponema, borrelia, leptospira, mycoplasma,ureaplasma, rickettsia, chlamydiae, candida, systemic mycoses,opportunistic mycoses, protozoa, nematodes, trematodes, cestodes,adenoviruses, herpesviruses (including, for example, herpes simplexvirus and Epstein Barr virus, and herpes zoster virus), poxviruses,papovaviruses, hepatitis viruses, (including, for example, hepatitis Bvirus and hepatitis C virus), papilloma viruses, orthomyxoviruses(including, for example, influenza A, influenza B, and influenza C),paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses,flaviviruses, bunyaviridae, rhabdoviruses, rotavirus, respiratorysyncitial virus, human immunodeficiency virus and retroviruses.Exemplary infectious diseases include but are not limited tocandidiasis, candidemia, aspergillosis, streptococcal pneumonia,streptococcal skin and oropharyngeal conditions, gram negative sepsis,tuberculosis, mononucleosis, influenza, respiratory illness caused byRespiratory Syncytial Virus, malaria, schistosomiasis, andtrypanosomiasis.

In one embodiment, the antibodies and fragments thereof provided hereinare useful in the treatment of diseases mediated by T-helper type 2(Th2) T cells, such as, for example, asthma, allergy, or graft versushost disease.

In one embodiment, the antibodies and fragments thereof provided hereinare useful in for the stimulation of an immune response in a subject inneed thereof. For example, in one embodiment, the anti-PD-1 antibodiesand fragments thereof may be administered in conjunction with an antigenof interest for the purpose of eliciting an immune response to saidantigen. An antigen of interest may be an antigen associated with apathogen such as a virus or bacterium. Thus, in one embodiment, thepresent invention provides a vaccine comprising an anti-PD-1 antibodyand an antigen, wherein the vaccine elicits an antigen-specific immuneresponse.

In one embodiment, the anti-PD-1 antibodies provided herein modulateregulatory T cell function. CD4+CD25+ regulatory T cells are lymphocytesthat suppress or reduce the effects of effector T cell functions. Theterms “regulatory T cell” and “Treg” are used interchangeably herein. Inone embodiment, the anti-PD-1 antibodies provided herein prevent orreverse the inhibitory effects of regulatory T cells on effector T cellcytokine production. For example, in one embodiment, the anti-PD-1antibodies provided herein restore the capacity for IFNγ production toeffector T cells in contact with regulatory T cells.

In one embodiment, the antibodies and fragments thereof disclosed hereinmay be administered to the subject by at least one route selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intratympanic,intrauterine, intravesical, intravitreal, bolus, subconjunctival,vaginal, rectal, buccal, sublingual, intranasal, intratumoral, andtransdermal.

In one embodiment, the antibodies and fragments thereof disclosed hereinmay be administered to a subject in need thereof in combination with oneor more additional therapeutic agent. In one embodiment, the antibodiesand fragments thereof may be administered to a subject before, during,and/or after administration to the subject of the additional therapeuticagent. In one embodiment, the additional therapeutic agent is achemotherapeutic agent, radiotherapeutic agent, cytokine, antibody orfragment thereof, or any other additional therapeutic that is indicatedfor the disease to be treated. In one embodiment, the anti-PD-1 antibodyand the additional therapeutic agent exhibit therapeutic synergy whenadministered together, whether concurrently or sequentially. In oneembodiment, the anti-PD-1 antibody and the additional therapeutic agentare administered in separate formulations. In another embodiment, theanti-PD-1 antibody and the additional therapeutic agent are administeredin the same formulation. In one embodiment, the anti-PD-1 antibodies andfragments provided herein enhance the immune modulating effect of theone or more additional therapeutic agent. In another embodiment, the oneor more additional therapeutic agent enhances the effect of theanti-PD-1 antibody or fragment thereof.

The present invention provides isolated antibodies and antigen bindingfragments thereof, and nucleic acids encoding such antibodies andfragments, as well as compositions comprising such isolated antibodies,fragments, and nucleic acids. The term “isolated” refers to a compoundof interest (e.g., an antibody or nucleic acid) that has been separatedfrom its natural environment. The present invention further providespharmaceutical compositions comprising the isolated antibodies orfragments thereof, or nucleic acids encoding such antibodies orfragments, and further comprising one or more pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers include, forexample, excipients, diluents, encapsulating materials, fillers,buffers, or other agents.

The use of the singular includes the plural unless specifically statedotherwise. The word “a” or “an” means “at least one” unless specificallystated otherwise. The use of “or” means “and/or” unless statedotherwise. The meaning of the phrase “at least one” is equivalent to themeaning of the phrase “one or more.” Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting. Also, terms such as “element” or “component” encompassboth elements or components comprising one unit and elements orcomponents comprising more than one unit unless specifically statedotherwise.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of non-critical parameters thatcould be changed or modified to yield essentially similar results.

EXAMPLES Example 1—Mouse Immunization and Production of Mouse AntibodiesAgainst Human PD-1

To generate antibodies against the human PD-1, cDNAs encoding the openreading frame of the extracellular domain of hPD-1 fused with ahistidine tag (hPD-1-HisTag, SEQ ID NO:1), mouse Fc (hPD-L1-mFc, SEQ IDNO:13), and human Fc tag (hPD-1-hFc, SEQ ID NO:5) were obtained by PCRand subcloned into expression vector pcDNA3.1 (Invitrogen CAT #:V-790),respectively. After transient expression in freestyle 293 cells,hPD-1-HisTag was purified with NTA column (GE healthcare), hPD-1-mFc andhPD-1-hFc were purified with Protein G column (GE healthcare).

To immunize mice necessary for generating hybridoma cell lines, 100 μgof human PD-1-mouse Fc fusion protein or and a complete Freund'sadjuvant in the same amount were mixed, and the mixture was administeredvia an subcutaneous injection to each of five 6 to 7-week-old BALB/cmice. After two weeks, the antigen (half the previously injected amount)was mixed with an incomplete Freund's adjuvant using the same method asdescribed above, and the mixture was administered to each mouse viasubcutaneous injection. After one week, final boosting was performed,and blood was collected from the tail of each mouse after three days toobtain serum. Then, serum was diluted at 1/1000 with PBS, and an ELISAwas performed to analyze whether the titer of the antibody recognizinghuman PD-1-mFc increased. Afterwards, mice in which a sufficient amountof the antibody was obtained were selected, and a cell fusion processwas performed on the selected mice.

Three days before a cell fusion experiment, a mixture of 50 μg of PBSand human PD-1-mFc fusion protein was administered via anintraperitoneal injection to each mouse. Each immunized mouse wasanesthetized, and its spleen located on the left side of the body wasthen extracted and ground with a mesh to isolate cells, which were mixedwith a culture medium (RPMI1640) to prepare a spleen cell suspension.The suspension was centrifuged to collect a cell layer. The obtained1×10⁸ of spleen cells were mixed with 1.5×10⁷ of myeloma cells (Sp2/0),and the mixture was centrifuged to precipitate the cells. Theprecipitate was slowly dispersed and treated with PEG Hybri-Max (SigmaInc., CAT #:7181). The mixed cells were distributed into 96-well platesat 0.1 ml per well and incubated at 37° C., 5% CO₂ incubator. On day 1,the cells were fed by the addition of an additional 0.1 ml mediacontaining serum and HAT plus 2× methotrexate for each well. On day 3and day 7, 0.1 ml of medium from each well was replaced with 0.1 ml offresh HT medium. The screening typically occurred between days 9-14.

Example 2—Selection of the Hybridoma Cells that Produce MonoclonalAntibodies Against Human PD-1 Protein Based on ELISA and FACS Analyses

ELISA binding analysis was conducted based on human PD-1-hFc Protein.96-well plates (Costar, Cat No:9018) were coated with 100 μL of 2 g/mlPD1-hFc (CrownBio) in coating buffer (PBS, Hyclone, Cat No:SH30256.01B)overnight at 4° C. The wells were aspirated and non-specific bindingsites were blocked by adding 200 μL of blocking buffer with 1% (w/v) ofbovine serum albumin (BSA, Roche, Cat No: 738328) and incubating for 1hour at 37° C. After the plates are washed three times with wash buffer(PBS with 0.05% (v/v) Tween20 (Sigma, Cat No:P1379), 100 μL/well of asuitable dilutions of hybridoma supernatant in blocking buffer wereadded and incubated at room temperature for 1 hour. The plates werewashed and incubated with 100 μL/well of Goat anti-Mouse IgG (H+L)(Thermo, Cat No: 31432) in blocking buffer for 60 min. After the plateswere washed, 100 μL/well of substrate solution (TMB(eBioscience, CatNo:00-4201-56) was added and the plates were incubated for 2 min at roomtemperature. 100 μL/well of stop solution (2N H2SO4) was added to stopthe reaction. The colorimetric signals were developed and read at 450 nmusing a Auto Plate SpectraMax Plus (Supplier: Molecular Devices; Model:MNR0643; Software: SoftMax Pro v5.4). Through this method, hybridomacell lines that produce antibodies highly specifically binding to thehuman PD-1 protein were repeatedly selected.

ELISA based ligand blockage analysis was conducted via blockingbiotinylated human PD-L1-mFcs from binding to human PD-1-hFc. PD-1-mFcantigen (CrownBio) was suspended in PBS (Hyclone, Cat No:SH30256.01B)buffer (2 ug/ml, 100 ul/well) and coated on the 96 well plate (costar,Cat No.:9018) 4° C. overnight. Plates were washed 3 times using washingbuffer: PBS+0.05% Tween 20(Sigma, Cat No.:P1379). 200 ul of blockingbuffer (PBS+1% BSA(Roche, Cat No.:738328)), was added to each well,incubated at 37° C. for 1 hour, and washed 3 times. Variousconcentrations (suitable dilutions of hybridoma supernatant in PBS) ofthe anti-PD-1 Abs were added to the wells (100 μl/well) and incubated at37° C. for 1 hour. Ligand was added (0.1 ug/ml PDL-1-mFc-biotin, 100μl/well), incubated at 37° C. for 2 h, and washed 3 times. Secondaryantibody (Avidin HRP eBioscience cat No.:E07418-1632, 1:500, 100ul/well) was added, incubated at 37° C. for 0.5 hour, and washed 3times. TMB (Sigma, Cat No.: T0440, 100 ul/well) was added, and incubatedfor 3 min at RT. To stop the reaction, 2N H2SO4 (100 ul/well), wasadded. The colorimetric signals were developed and read at 450 nm usinga Auto Plate SpectraMax Plus (Supplier: Molecular Devices; Model:MNR0643; Software: SoftMax Pro v5.4).

Cell binding analysis of antibodies was performed based on hPD-1-293Tcell line. 2×10⁵ 293T-PD-1 cells were used for each reaction by puttingthem into each well of 96-well culture plates. The cells were incubatedwith the indicated antibody (20 ug/ml with the dilution of 1/5) at 4° C.for 1 h. Cells were washed three times with FACS buffer. A secondaryantibody (PE Goat anti-mouse: 1:200; PE mouse anti-human: 1:10) wasadded to the cells at 100 ul/well, and incubated at 4° C. for 40 min.Cells were washed three times with FACS buffer and analyzed by FACSArray.

FACS based ligand blockage analysis was conducted to determine theanti-PD-1 hybridoma antibodies in the blockage of biotinylated humanPD-L1 and PD-L2 binding to hPD-1-293T cells using a flow cytometryassay. PD-1 expressing 293T cells were suspended in FACS buffer (PBSwith 3% fetal calf serum). Various concentrations of the testinghybridoma antibodies were added to the cell suspension and incubated at4° C. for 60 minutes in 96 well plates. Biotin-labeled PD-L1 protein orBiotin-labeled PD-L2 protein was added into the wells and incubated at4° C. for 60 minutes. Plates were washed 3 times, and mouse anti-biotinPE antibody (Biolgend, cat #409004) was added. Flow cytometric analyseswere performed using a FACS Array. The results of the study are depictedin FIG. 1A (PD-L1) and FIG. 1B (PD-L2). The anti-PD-1 monoclonalantibodies blocked binding of PD-L1 or PD-L2 to 293T cells transfectedwith human PD-1, as measured by the mean fluorescent intensity (MFI) ofstaining. These data demonstrated that the anti-PD-1 antibodies blockbinding of ligand PD-L1 and PD-L2 to cell surface PD-1.

Example 3—Subcloning to Obtain Monoclonal Antibody Clones andPurification of Anti-hPD-1 Antibodies

Subcloning is based on the procedure of limited dilution, and isdesigned to obtain individual hybridoma clones producing monoclonalantibodies. Each of the hybridomas was subjected to multiple rounds (4rounds) of limiting dilution. For each round of subcloning, the cloneswere tested by ELISA and FACS based blockage analyses.

Antibody purification was conducted for a total of twenty two anti-hPD-1hybridoma antibodies. The hybridoma cells were cultured in Dulbecco'sModified Eagle's medium (GIBCO; Invitrogen Corporation, Carlsbad,Calif.) containing 10% fetal calf serum, 1% penicillin/streptomycin, 2%L-glutamine, and 1% adjusted NaHCO₃ solution. The selected hybridomacells were then adapted in serum free culture medium and the antibodywas purified from the supernatant using Protein-G column (GEhealthcare). After washing with PBS, bound antibodies were eluted using0.1 M Glycine pH3.0, followed by pH neutralization using 2.0 M Tris.Ultra-15 centrifugal concentrators (Amicon) were used for bufferexchanging and antibody concentrating.

Example 4—Characterization of the Purified Murine Anti-hPD-1 Antibodiesin Binding and Ligand Blockage Activities Based on ELISA and FACSAnalyses

The purified hybridoma antibodies were characterized further based onELISA and FACS analyses. The methods used were similar to thosedescribed above in Example 2 except that in these cases, purifiedantibodies were measured in amount and concentration, and the resultswere used to calculate EC50 and IC50 values. The following tables,Tables 1-5, show the results of 10 antibodies.

TABLE 1 ELISA based binding EC50 of 10 murine anti-PD-1 antibodies ng/ml15H5 22A5 14A6 6E1 7D3 10D1 4C10 13F1 5A8 7A4 EC50 13.35 19.38 7.0715.67 24.36 17.18 13.25 17.33 11.25 9.32

TABLE 2 ELISA based blockage IC50 of 10 murine anti-PD-1 antibodiesng/ml 15H5 22A5 14A6 6E1 7D3 10D1 4C10 13F1 5A8 7A4 IC50 873.2 1114.8923 961.2 982.0 1409 1464.3 701.0 1128.7 698.8

TABLE 3 FACS based binding EC50 of 10 murine anti-PD-1 antibodies ng/ml15H5 22A5 14A6 6.00E+01 7D3 10D1 4C10 13F1 5A8 7A4 EC50 45.37 77.45 NA49.09 52.66 NA 108.9 30.79 NA 38.93

TABLE 4 FACS based PD-L1 blockage IC50 of 10 murine anti-PD-1 antibodiesselected. ng/ml 15H5 22A5 14A6 6E1 7D3 10D1 4C10 13F1 5A8 7A4 IC50 35.6456.22 21.13 26.85 38 36.9 83.32 19.86 28.22 28.85

TABLE 5 FACS based PD-L2 blockage IC50 of 10 murine anti-PD-1 antibodiesselected ng/ml 15H5 22A5 14A6 6E1 7D3 10D1 4C10 13F1 5A8 7A4 IC50 63.04210.1 91.98 113.7 140.5 150 250.7 52.93 18.84 4.45

Example 5: Biacore Analysis of the Murine Anti-PD-1 Antibodies

To further characterize the binding characteristics of the antibodies,10 hybridoma antibodies were profiled using Biacore (Biacore 3000, GE)to elucidate binding kinetics and calculate equilibrium bindingconstants. This assay was performed by capture method, using the mouseantibody capture kit (BR-1008-38, GE). After diluting anti-mouse Fc mabto 25 μg/ml in pH 5.0 immobilization buffer, immobilization wasconducted with the parameters shown in Table 6 at a flow rate of 5μl/min. The kinetic runs were done by 1) injecting ligand for typical0.5-1 min at flow rate of 10 μl/min.; 2) injecting analytes of choicefor typical 3 min followed by dissociation in running buffer (1×PBS-P20)for typical 5-10 min at flow rate of 30 μl/min.; and 3) injectingregeneration solution 10 mM Glycine pH1.7 for typical 1-2 min at flowrate of 10 μl/min.

TABLE 6 Biacore parameters. Event Injection Conditions ActivationEDC/NHS (1:1 7 minutes Mix) Immobilization Diluted Anti- 4 minutes toachieve human Fc mAb ~7000 RU Immobilization level DeactivationEthanolamine-HCl 7 minutes

The results of the study are shown in Table 7. Each of the anti-humanPD1 antibodies exhibited an association rate (ka) in the range of1.11E+05 l/Ms to 8.40E+05 l/Ms; a dissociation rate (kd) in the range of2.83E-05 l/s to 7.55E-05 l/s; an equilibrium association constant (KA)in the range of 1.60E+10 l/M to 5.44E+10 l/M; and an affinity (KD) inthe range of 1.84E-11 M to 6.23E-11 M (0.0184 nM to 0.0623 nM).

TABLE 7 KD values of anti-PD-1 hybridoma antibodies. ka (1/Ms) kd (1/s)KA (1/M) KD ( M) 10D1 8.40E+05 5.24E−05 1.60E+10 6.23E−11 14A6 1.51E+064.52E−05 3.33E+10 3.00E−11 22A5 1.49E+06 2.88E−05 5.17E+10 1.93E−11 4C107.91E+05 2.63E−05 3.01E+10 3.32E−11 7A4 1.96E+06 4.82E−05 4.06E+102.46E−11 6E1 1.11E+06 2.83E−05 3.92E+10 2.55E−11 13F1 1.41E+06 3.92E−053.60E+10 2.78E−11 15H5 2.00E+06 3.67E−05 5.44E+10 1.84E−11 5A8 1.29E+067.55E−05 1.70E+10 5.87E−11 7D3 1.14E+06 2.83E−05 4.02E+10 2.49E−11

Example 6: Cross-Reactivity Among Species and Among Similar Molecules

To assess the species cross-reactivity of the antibodies, the mouse andcynomolgus macaque PD-1 receptors were cloned by PCR and stablytransfected 293T-PD-1 cells were generated. The antibodies were testedfor binding to the cynomolgus receptor using protein based ELISA. Theresults of the study showed that the antibodies bind with equal affinityto human and cynomolgus PD-1 and block binding of hPD-L1/Fc andhPD-L2/Fc to cynomolgous PD-1 with similar efficacy as compared to humanPD-1. None of the antibodies selected bound mouse PD-1 with detectableaffinity in any of the assays used. None cross reacts with human CTLA4,ICOS and CD28 (see Table 8).

Example 7: Effect of the Anti-PD-1 Hybridoma Antibodies on CytokineProduction in a Mixed Lymphocyte Reaction

A mixed lymphocyte reaction was used to demonstrate the effect ofblocking the PD-1 pathway on lymphocyte effector cells. T cells in theassay were tested for proliferation, IFN-γ secretion and IL-2 secretionin the presence or absence of a murine anti-human PD-1 monoclonalantibody. In the assay, human CD4+ T-cells were purified from PBMC usinga CD4+ negative selection (Miltenyi Biotech, cat #130-091-155). MatureDendritic cells (DC) were derived from purified monocytes (Miltenyi,Mo-DC Generation Toolbox, cat #130-093-568) culture with Mo-DCDifferentiation Medium for 7 days; then, DC maturation was induced withMo-Dc Maturation for 2 days. Each culture contained 10⁵ purified T-cellsand 10⁴ allogeneic dendritic cells in a total volume of 200 μl.Anti-PD-1 monoclonal antibody 4C10, 5A8, 6E1, 7D3, 7A4, 10D1, 13F1,14A6, 15H5, or 22A5 was added to each culture at different antibodyconcentrations. Either no antibody or an isotype control antibody wasused as a negative control. The cells were cultured for 5 days at 37° C.On 5 day, 50 μl of medium was collected for measurement of IL-2 andIFN-γ. The levels of IFN-γ and IL-2 in the culture fluid were measuredusing an EIA hIFN-γ ELISA kit (R&D, cat #DY285) and IL-2 ELISA kit(eBioscience) The results of the study are provided in FIG. 2 (IL-2secretion) and FIG. 3 (IFN-γ secretion) and show that the anti-humanPD-1 monoclonal antibodies promoted T-cell proliferation, IFN-γsecretion and IL-2 secretion in a concentration dependent manner. Incontrast, cultures containing the isotype control antibody did not showan increase in T cell proliferation, IFN-γ or IL-2 secretion.

Example 8: Features of 10 Murine Anti-hPD-1 Antibodies

Characteristics of 10 anti-PD1 monoclonal antibodies that were purifiedand characterized are summarized in Table 8. These antibodies boundtightly to PD-1 (with dissociation constants in the 20 uM to 3 nM range)and were capable of blocking the interaction with both PD-L1 and PD-L2with varying IC50 values. Each of the antibodies induced IL2 and IFNγproduction. None of the 10 antibodies crossreacted with CTLA4, ICOS, orCD28. Each of the antibodies bound cynomolgous PD-1. Each of theantibodies, when added in solution acted as receptor antagonists,ultimately enhanced T cell responses (see Example 5).

TABLE 8 Summary of characterized features of 10 Murine anti-hPD-1antibodies. PD-L1 PD-L2 Interaction ELISA FACS Blockage BlockageBlockage with Binding at ELISA FACS FACS Tcell Tcell CTLA4, InteractionSelected KD EC50 20 EC50 EC50 EC50 activation activation ICOS with cyno-No. Abs (Biacore) (ng/ml) ug/ml (ug/ml) (ng/ml) (ng/ml) IL2 IFN-g CD28PD-1 1 15H5 1.84E−11 13.35 +++ 873.2 35.64 63.04 +++ ++ − +++ 2 22A51.93E−11 19.38 +++ 1114.8 56.22 210.10 +++ ++ − +++ 3 14A6 3.00E−11 7.07+++ 923 21.13 91.98 +++ +++ − +++ 4 6E1 2.55E−11 15.67 +++ 961.2 26.85113.70 +++ ++ − +++ 5 7D3 2.49E−11 24.36 +++ 982 38 140.50 ++ +++ − +++6 10D1 6.23E−11 17.18 +++ 1409 36.9 150.00 +++ +++ − +++ 7 4C10 3.32E−1113.25 +++ 1464.3 83.32 250.70 +++ +++ − +++ 8 13F1 2.78E−11 17.33 +++701 19.86 52.93 +++ ++++ − +++ 9 5A8 5.87E−11 11.25 +++ 1128.7 28.2218.84 +++ +++ − +++ 10 7A4 NA 9.32 +++ 698.8 28.85 4.45 ++++ +++ − +++

Example 9: Anti-PD-1 Antibody cDNA Sequences Cloning and Humanization

Cloning of Immunoglobulin cDNAs

Total RNA isolated from the hybridoma cell line producing hPD-1 antibodyby RNeasy Mini Kit (Qiagen, CAT #:74104) was used as the template tosynthesize first-strand cDNA with SuperScript® II Reverse Transcriptase(Life Technology, CAT #:18064-14) according to the manufacturer'sinstructions. The cDNA product was then subjected to PCR in a 50 μlvolume reaction mixture using degenerate mouse IgG primers(Kettleborough Calif., et al, European Journal of Immunology 23: 206-211(1993), Strebe N, et al, Antibody Engineering 1:3-14 (2010)). Thereaction was carried out in a S1000™ Thermal Cycler (Bio-Rad, CAT#:184-2000) with 30 cycles of: 94° C., 1.5 minutes for denaturation; 50°C., 1 minutes for annealing; and 72° C., 1 minute for synthesis. At theend of the 30th cycle, the reaction mixture was incubated another 7minutes at 72° C. for extension.

The PCR mixture was subjected to electrophoresis in a 1%agarose/Tris-Borate gel containing 0.5 μg/ml ethidium bromide. DNAfragments having the expected sizes (approximately 400 bp for the heavychain and the light chain) were excised from the gel and purified. 3 μlof purified PCR product were cloned into the pMD-18T vector (Takara, CAT#:D101A) and transformed into One Shot® TOP10 Chemically Competent E.coli (Invitrogen, CAT #:C4040-03). Clones were screened by colony PCRusing universal M13 forward and reverse primers, and 10 positive clonesfrom each reaction were chosen for DNA sequencing in both directionsusing M13 forward and M13 reverse primers.

The variable region sequences of antibodies 4C10 (SEQ ID NOs: 28, 33),5A8 (SEQ ID NOs: 99, 104), 6E1 (SEQ ID NOs: 89, 94), 7D3 (SEQ ID NOs:39, 44), 7A4 (SEQ ID NOs: 109, 114), 10D1 (SEQ ID NOs: 18, 23), 13F1(SEQ ID NOs: 49, 54), 14A6 (SEQ ID NOs:69, 74), 15H5 (SEQ ID NOs: 59,64) and 22A5 (SEQ ID NOs: 79, 84) were amplified from the correspondinghybridoma clones. These antibodies showed desired functions, such asblocking PD-1 binding to PD-L1 and enhanced T cell activation andcytokine release.

Construction and Expression of Chimeric 7A4 and 13F1 Antibody

7A4 and 13F1 chimeric light chains (SEQ ID NOs: 123 and 129,respectively) were constructed by linking the PCR-cloned cDNAs of mouseVL regions to human kappa and IgG1, respectively. 7A4 and 13F1 chimericIgG1 heavy chains (SEQ ID NOs: 119 and 125, respectively) wereconstructed by linking the PCR-cloned cDNAs of mouse VH regions to humanIgG1 constant region. 7A4 and 13F1 chimeric IgG4 heavy chains (SEQ IDNOs: 121 and 127, respectively) were constructed by linking thePCR-cloned cDNAs of mouse VH regions to human IgG4 constant region The5′ends of the mouse cDNA sequences were modified using PCR primersdesigned to add a leader sequence to both light chain and heavy chain.

Freestyle 293 cells (200 mL at 10⁶/mL) were transfected with 100 μg ofeach of the chimeric heavy and light chain expression plasmids andcultured for 6 days. The chimeric antibody in the supernatant was thenpurified with Protein-G column (GE healthcare). Binding of the chimericantibodies to PD-1 was measured by ELISA and Biacore as described abovein Examples 2 and 5, and was shown to bind to PD-1 with comparableaffinity to that of the murine parent antibody. Table 9 shows thebinding EC50 of each of the chimeric anti-PD-1 antibodies as measured byELISA. Table 10 shows the PD-L1 blockage IC50 of each of the chimericanti-PD-1 antibodies as measured by ELISA. Table 11 shows the bindingEC50 of each of the chimeric anti-PD-1 antibodies as measured by FACS.Table 12 shows the PD-L1 blockage IC50 of each of the chimeric anti-PD-1antibodies as measured by FACS.

TABLE 9 ELISA based binding EC50 of chimeric anti-PD-1 antibodies ng/ml15H5 22A5 14A6 6E1 7D3 10D1 4C10 13F1 7A4 hIgG4 hIgG4 hIgG4 hIgG4 hIgG4hIgG4 hIgG4 hIgG4 hIgG4 EC50 (ug/ml) 81.8 41.3 64.6 32.54 51.7 58.894.56 58.73 62

TABLE 10 ELISA based blockage IC50 of chimeric anti-PD-1 antibodiesng/ml 15H5 22A5 14A6 6E1 7D3 10D1 4C10 13F1 7A4 hIgG4 hIgG4 hIgG4 hIgG4hIgG4 hIgG4 hIgG4 hIgG4 hIgG4 IC50 1367.0 1010.9 823.4 868.6 948.11034.5 977.6 856.2 871.1

TABLE 11 FACS based binding EC50 of chimeric anti-PD-1 antibodies ng/ml15H5 22A5 14A6 6E1 7D3 10D1 4C10 13F1 7A4 hIgG4 hIgG4 hIgG4 hIgG4 hIgG4hIgG4 hIgG4 hIgG4 hIgG4 EC50 243.6 145.3 103.4 143.1 130.9 218.8 220.6113.1 91.6

TABLE 12 FACS based PD-L1 blockage IC50 of chimeric anti-PD-1 antibodiesng/ml 15H5 22A5 14A6 6E1 7D3 10D1 4C10 13F1 7A4 hIgG4 hIgG4 hIgG4 hIgG4hIgG4 hIgG4 hIgG4 hIgG4 hIgG4 IC50 100.5 84.26 61.37 54.01 NA 40.33 12952.13 70.55

Mixed lympohcyte reactions as described above in Example 7 were used todetermine the effect of the chimeric anti-PD-1 antibodies on IL-2secretion (FIG. 4 ) and IFN-γ secretion (FIG. 5 ) from T cells. Each ofthe chimeric anti-PD-1 monoclonal antibodies promoted IL-2 secretion andIFNγ secretion in a concentration dependent manner in the MLR assay. Incontrast, the isotype control antibody (hIgG4) did not elicit IL-2secretion or IFNγ secretion at any concentration tested.

Antibody Humanization Design

7A4 and 13F1 antibody were humanized using a CDR grafting approach (U.S.Pat. No. 5,225,539, incorporated herein by reference in its entirety).The light chain and heavy chain variable chain sequences of the murineantibody 7A4 and 13F1 were compared to those available in the ResearchCollaboratory for Structural Bioinformatics (RCSB) protein databank bysearching the NCBI database,http://www.ncbi.nlm.nih.gov/igblast/igblast.cgi. The model of 7A4 and13F1 were generated respectively based on the VH and VL structure withthe highest sequence homology.

The template human antibodies to be grafted with the complementarydetermining regions (CDRs) in the VH and VL of mouse 7A4 and 13F1antibody were selected from human antibody germlines having an aminoacid sequence with high homology with the mouse 7A4 and 13F1 antibody bysearching the IMGT/Domain Gap Align 3D structure database,http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi. For 7A4, thetemplate human VH selected was a combination of IGHV2-5*10 and IGHJ4*01,and template human VL selected was a combination of IGKV1-33*01 andIGKJ2*01. For 13F1, the template human VH selected was a combination ofIGHV3-21*04 and IGHJ4*01, and template human VL selected was acombination of IGKV7-3*01 and IGKJ2*01.

CDR amino acid sequences of the aforementioned template human antibodieswere substituted by the amino acid sequence of CDRs of mouse 7A4 and13F1 antibody. In addition, the frameworks of the above-mentionedtemplate human antibody VH and VL were grafted with the necessary aminoacid sequences from VH and VL of mouse 7A4 and 13F1 antibody to give afunctional humanized antibody. As for VH and VL of 7A4 and 13F1, severalsites of framework amino acid of the aforementioned template humanantibody were back mutated to the corresponding amino acid sequences inmouse 7A4 and 13F1 antibody. For the light chain variable region ofhumanized 7A4 antibody, the amino acid at position 40 was mutated fromTyr (Y) to Phe (F) and the amino acid at position 72 was mutated fromGly (G) to Arg (R); and for the heavy chain variable region of humanized7A4 antibody, the amino acid at position 2 was mutated from Val (V) toIle (I), the amino acid at position 46 was mutated from Glu (E) to Lys(K), and the amino acid at position 70 was mutated from Phe (F) to Ile(I). For the light chain variable region of humanized 13F1 antibody, theamino acid at position 45 was mutated from Leu (L) to Pro (P) and theamino acid at position 70 was mutated from Phe (F) to Tyr (Y); and forthe heavy chain variable region of humanized 13F1 antibody, the aminoacid at position 26 was mutated from Gly (G) to Tyr (Y), the amino acidat position 48 was mutated from Ile (I) to Met (M), the amino acid atposition 49 was mutated from Gly (G) to Ala (A), the amino acid atposition 67 was mutated from Val (V) to Ile (I), and the amino acid atposition 71 was mutated from Val (V) to Arg (R).

The amino acid sequences of the variable light and variable heavy chainsof humanized 13F1 antibody were designated SEQ ID NOs: 143 and 141,respectively. The base sequences of DNAs encoding the amino acidsequences were designed (SEQ ID NO: 140 and 142, respectively). Theamino acid sequences of the variable light and variable heavy chains ofhumanized 7A4 antibody were designated SEQ ID NOs: 133 and 131,respectively. The base sequences of DNAs encoding the amino acidsequences were designed (SEQ ID NO: 130 and 132, respectively).

IgG1 and IgG4 versions of the humanized 7A4 and 13F1 antibodies wereproduced (h13F1-IgG1, h13F1-IgG4, h7A4-IgG1 and h7A4-IgG4). The IgG1constant region carries D265A mutation (Clynes R, et al, Nature Medicine6: 443-446 (2000)) while IgG4 constant region has F234A and L235A doublemutation (Xu D, et al, Cellular Immunology 200: 16-26 (2000)). Theconstant region sequences are disclosed in SEQ ID NOS: 150 and 151. Thefull light and heavy chain amino acid sequences for h13F1-IgG1 (SEQ IDNOs: 149 and 145), h13F1-IgG4 (SEQ ID NOs: 149 and 147), h7A4-IgG1 (SEQID NOs: 139 and 135), and h7A4-IgG4 (SEQ ID NOs: 139 and 137) areprovided above in Table 3. To remove the potential deamidation site inthe light chain of 7A4, Asn85 is mutated to Asp (h7A4D). The light chainvariable region (SEQ ID NO: 152) and full light amino acid sequences(SEQ ID NO: 153) are also provided above in Table 3.

Construction and Expression of Humanized 7A4, 7A4D and 13F1 Antibodies

DNA encoding humanized 7A4, 7A4D and 13F1 antibody light chain and heavychain was synthesized and cloned to the expression vector pcDNA3.1(Invitrogen, CAT: #V-790). Freestyle 293 cells (200 mL at 10⁶/mL) weretransfected with 100 μg of each of the humanized heavy and light chainexpression plasmids and cultured for 6 days. The humanized antibody inthe supernatant was then purified with Protein-G column (GE healthcare).

Example 10: Characterization of Humanized Anti-PD-1 Antibodies inBinding Activity and Specificity, and Ligand (PD-L1) Blockage Activity

After generation and purification of humanized 13F1-hIgG1, 13F1-hIgG4,7A4-IgG1 and 7A4-hIgG4 antibodies, the binding and specificity of theantibodies were determined based on ELISA-based binding and PD-1blockage analyses, as well as FACS-based binding and PD-L1 blockageanalyses. The methods used were similar to those described above inExamples 2 and 4.

In the ELISA-based binding assays, humanized 13F1 antibodieshu-13F1-hIgG1 and hu-13F1-hIgG4 exhibited similar binding to PD-1compared to the chimeric antibody 13F1-chimeric (FIG. 6A, top panel);and humanized 7A4 antibodies hu-7A4-D265A-hIgG1 and 7A4-huIgG4 exhibitedsimilar binding to PD-1 compared to the chimeric 7A4 antibodies (FIG.6B, top panel). In contrast, the isotype control hIgG4 antibody did notexhibit PD-1 binding. The bottom panels of FIG. 6A and FIG. 6B show theEC50 for each of the antibodies tested, calculated from the ELISAbinding data, and demonstrates that the humanized 13F1 and 7A4antibodies exhibited PD-1 binding.

Similarly, in the FACS-based binding assays, humanized 13F1 antibodieshu-13F1-hIgG1 and hu-13F1-hIgG4 (FIG. 7A, top panel) and humanized 7A4antibodies hu-7A4-D265A-hIgG1 and 7A4-huIgG4 (FIG. 7B, top panel)exhibited binding to PD-1. The EC50 calculated from the FACS bindingdata for humanized 13F1 and 7A4 antibodies are shown in FIG. 7A and FIG.7B, respectively.

FIG. 8 shows the results of the ELISA-based ligand blocking assays forhumanized 13F1 and humanized 7A4 antibodies. As shown in FIG. 8A andFIG. 8B, the humanized 13F1 and 7A4 antibodies, respectively, exhibitedsimilar ligand blockage activity relative to the corresponding chimericantibody. Quantification of the IC50 for each of the humanized andchimeric antibodies is shown in FIG. 8C.

FIG. 9 shows that each of the humanized 13F1 and humanized 7A4antibodies blocked PD-L1 binding as measured by FACS-based ligandblockage assay. The bottom panel of FIG. 9 provides the IC50 for each ofthe humanized antibodies.

Example 11: Biacore Kinetic Analysis of the Humanized 13F1, 7A4 and 7A4DAnti-PD-1 Antibodies

To characterize the binding characteristics of the humanized antibodies,the binding kinetics between PD-1 and PD-1 antibodies were measured byBiacore3000 and recorded with a data collection rate of 1 Hz. Thepolyclonal rabbit anti-mouse IgG (GE, BR-1008-38) was diluted with 10 mMpH 5.0 sodium acetate and immobilized onto reference and experiment flowcells of a CM5 biosensor chip to around 15000 RU using an amine couplingkit (GE, BR10050). In the beginning of each cycle, diluted test antibody(1.5 μg/mL) was injected over experiment flow cell for 1 minute to becaptured. PD-1 analyte series were prepared by diluting the stocks withrunning buffer to 100 nM followed by 2× serial dilution in the samebuffer down to 0.78 nM. Analytes were injected in series over thereference and experiment flow cells for 3 minutes at a flow rate of 30μL/minute. Then running buffer (PBS with 0.05% P20) was allowed to flowover for 10 minutes at a flow rate of 30 μL/minute. At the end of eachcycle, the biosensor surface was regenerated with 3 minutes injection of10 mM pH1.7 Glycine-HCl buffer at a flow rate of 10 μL/minute. For eachanalyte sample injection (i.e. each cycle), binding responses obtainedfrom the experimental biosensor surface were double referenced bysubtracting simultaneously recorded responses from the reference surfacefollowed by additional subtraction of responses from a single referencedrunning buffer sample. The association and dissociation rate constants(ka and kd) were determined simultaneously by fitting double-referencedsensorgrams of the entire titration series to Langmuir model (1.1) usingBiaevaluation 4.0 software. The dissociation constant, KD, wascalculated from the determined rate constants by the relation KD=kd/ka.As shown in FIGS. 10 and 17A, the humanized anti-PD-1 antibodies 13F1,7A4, and 7A4D bound human PD-1 with high affinity. The Biocore bindingcurves are shown in FIGS. 10 and 17A, top panel, and the quantifiedbinding data are summarized in FIGS. 10 and 17A, bottom panel. FIG. 17Bindicates the blockage IC50 of PD-L1's binding to 293T-PD1 cells by7A4D-hIgG4 antibody.

Example 12: Effect of Humanized Anti-PD-1 Antibodies on CytokineProduction in a Mixed Lymphocyte Reaction (MLR)

Mixed Lymphocyte Reactions (MLR) were employed to demonstrate theability of the humanized antibodies to block the PD-1 pathway inlymphocyte effector cells. T cells in the assay were tested for IFNγ andIL-2 secretion in the presence or absence of anti-PD-1 antibodies. HumanCD⁴⁺ T-cells were purified from human PBMC using a CD4 negativeselection isolation kit (Mitenyi Biotech, cat #130-091-155). Immaturedendritic cells (DC) were derived from monocytes isolated from humanPBMC using the Mo-DC Generation Toolbox (Miltenyi, cat #130-093-568).The cells were cultured with Mo-DC Differentiation Medium for 7 days,and were then induced to be mature DC with Mo-Dc Maturation medium for 2days. To set up the MLR, 10⁵ purified T-cells and 10⁴ allogeneic matureDC cells in a total volume of 200 were added to each well. The testingantibody was assayed at a range of concentrations from 20 μg/ml to 0.002μg/ml. Either no antibody or an isotype control antibody (hIgG4) wasused as a negative control. The cells were cultured for 5 days at 37° C.On day 6^(th), the levels of IFN-γ and IL-2 in the culture medium weremeasured using the IL-2 ELISA kit (eBioscience) and hIFN-γ ELISA kit(R&D, cat #DY285). For humanized 13F1 antibodies, the results are shownin FIG. 11 (IL-2 production) and FIG. 12 (IFNγ production). Each of thehumanized 13F1 antibodies promoted IL-2 and IFNγ production in aconcentration dependent manner. Similarly, humanized 7A4 and 7A4Dantibodies promoted IL-2 (FIGS. 13 and 18 ) and IFNγ (FIGS. 14 and 19 )production in a concentration dependent manner. Cultures containing theisotype control antibody did not show increase in IFN-γ and IL-2secretion. Thus, the results of the study showed that the humanized PD-1antibodies block the PD-1 pathway, stimulating T cell immune responses.

Example 13: Human Recall T Cell Response to Tetanus Toxoid Challenge isEnhanced by Humanized Anti-PD-1 Antibody

To investigate whether the antigen-specific T cell receptor triggeringwas modulated by blocking PD-1/PD-L1 pathway with anti-PD-1 antibodies,the human T-cell recall assay was employed using tetanus toxoid (TT)antigen to stimulate pre-existing memory T cells in the blood of healthyTT immunized donors. To this end, fresh PBMC recently collected (samplescollected within less than 1 year) from TT immunized donors were platedinto 96-well round bottom plates (costar, cat #3799) at 4×10⁵ cells/wellusing RPMI1640 (Invitrogen, cat #A10491-01) supplemented with 80 U/mlpenicillin, 80 g/ml streptomycin and 30% autologous serum. Humanized13F1 or 7A4 antibodies were added at various concentrations, andstimulated with 0.1 ug/ml SEB and 1 ug/ml TT (Astarte Biologies). Afterco-culture for 7 days at 37° C., 5% CO₂, the supernatant was harvestedand the concentration of IFN-γ was measured. The results of the studyare shown in FIG. 15 , and demonstrate that, compared to TT antigenalone, PD-L1 blockage with anti-PD-1 antibodies resulted in enhancedIFN-γ secretion by memory T cells.

Example 14: Effect of Humanized Anti-PD-1 Antibody on Autologous T CellActivation

In this example, the effect of blocking PD-/PD-L1 pathway by humanizedanti-PD-1 antibody on T cell activation was examined. Purified humanCD4+ T cells (Mitenyi Biotech, cat #130-091-155) were activated with 1μg/ml soluble anti-CD3 antibody (R&D, cat #MAB100) in the presence ofautologous monocyte-derived dendritic cells (DCs). After three days ofactivation in the presence or absence of titrated anti-PD-1 antibody,culture medium was harvested and the concentration of IFNγ was measuredwith ELISA. The results are shown in FIG. 16 and indicate that PD-L1blockage by anti-PD-1 antibody enhanced IFN-γ secretion by T cells.

The invention claimed is:
 1. A method for treating an infectious diseasein a subject in need thereof, the method comprising administering to thesubject an antibody or fragment thereof that binds to PD-1, wherein theantibody or fragment thereof comprises: (i) a light chain CDR1, CDR2,and CDR3 sequence comprising SEQ ID NO: 115, 116, and 117, respectively,and a heavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:110, 111, and 112, respectively; (ii) a light chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 55, 56, and 57, respectively, and a heavychain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 50, 51, and52, respectively (iii) a light chain CDR1, CDR2, and CDR3 sequencecomprising SEQ ID NO: 24, 25, and 26, respectively, and a heavy chainCDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 19, 20, and 21,respectively; (iv) a light chain CDR1, CDR2, and CDR3 sequencecomprising SEQ ID NO: 34, 35, and 36, respectively, and a heavy chainCDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 29, 30, and 31,respectively; (v) a light chain CDR1, CDR2, and CDR3 sequence comprisingSEQ ID NO: 45, 46, and 47, respectively, and a heavy chain CDR1, CDR2,and CDR3 sequence comprising SEQ ID NO: 40, 41, and 42, respectively;(vi) a light chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:65, 66, and 67, respectively, and a heavy chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 60, 61, and 62, respectively; (vii) alight chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 75, 76,and 77, respectively, and a heavy chain CDR1, CDR2, and CDR3 sequencecomprising SEQ ID NO: 70, 71, and 72, respectively; (viii) a light chainCDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 85, 86, and 87,respectively, and a heavy chain CDR1, CDR2, and CDR3 sequence comprisingSEQ ID NO: 80, 81, and 82, respectively; (ix) a light chain CDR1, CDR2,and CDR3 sequence comprising SEQ ID NO: 95, 96, and 97, respectively,and a heavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO:90, 91, and 92, respectively; or (x) a light chain CDR1, CDR2, and CDR3sequence comprising SEQ ID NO: 105, 106, and 107, respectively, and aheavy chain CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 100,101, and 102, respectively.
 2. The method of claim 1, wherein theantibody or fragment thereof comprises a light chain CDR1, CDR2, andCDR3 comprising an amino acid sequence according to SEQ ID NOs: 115,116, and 117, respectively; and a heavy chain CDR1, CDR2, and CDR3comprising an amino acid sequence according to SEQ ID NOs: 110, 111, and112, respectively.
 3. The method of claim 1, wherein the antibody orfragment thereof comprises a light chain CDR1, CDR2, and CDR3 comprisingan amino acid sequence according to SEQ ID NOs: 55, 56, and 57,respectively; and a heavy chain CDR1, CDR2, and CDR3 comprising an aminoacid sequence according to SEQ ID NOs: 50, 51, and 52, respectively. 4.The method of claim 1, wherein the antibody or fragment thereof ishumanized.
 5. The method of claim 1, wherein the antibody or fragmentthereof comprises (i) a light chain variable region comprising SEQ IDNO: 33 and a heavy chain variable region comprising SEQ ID NO: 28; (ii)a light chain variable region comprising SEQ ID NO: 54 and a heavy chainvariable region comprising SEQ ID NO: 49; (iii) a light chain variableregion comprising SEQ ID NO: 64 and a heavy chain variable regioncomprising SEQ ID NO: 59; (iv) a light chain variable region comprisingSEQ ID NO: 104 and a heavy chain variable region comprising SEQ ID NO:99; (v) a light chain variable region comprising SEQ ID NO: 114 and aheavy chain variable region comprising SEQ ID NO: 109; (vi) a lightchain variable region comprising SEQ ID NO: 143 and a heavy chainvariable region comprising SEQ ID NO: 141; or (vii) a light chainvariable region comprising SEQ ID NO: 152 and a heavy chain variableregion comprising SEQ ID NO:
 131. 6. The method of claim 1, wherein theantibody or fragment thereof comprises a light chain variable regionaccording to SEQ ID NO: 133 or 152 and a heavy chain variable regionaccording to SEQ ID NO:
 131. 7. The method of claim 1, wherein theantibody or fragment thereof comprises a light chain variable regionaccording to SEQ ID NO: 143 and a heavy chain variable region accordingto SEQ ID NO:
 141. 8. The method of claim 1, wherein the antibody orfragment thereof is selected from the group consisting of a monoclonalantibody, an scFv, a Fab fragment, an Fab′ fragment, and an F(ab)′fragment.
 9. The method of claim 1, wherein the method further comprisesadministering the antibody or fragment thereof to the subject incombination with an additional therapeutic agent.
 10. The method ofclaim 1, wherein the infectious disease is caused by a bacterial orviral agent.
 11. The method of claim 1, wherein the infectious diseaseis associated with PD-L1 expression on infected cells and/or with PD-1expression on T cells.
 12. The method of claim 1, wherein the infectiousdisease is associated with a chronic infection.
 13. The method of claim1, wherein the infectious disease is selected from the group consistingof candidiasis, candidemia, aspergillosis, streptococcal pneumonia,streptococcal skin and oropharyngeal conditions, gram negative sepsis,tuberculosis, mononucleosis, influenza, respiratory illness caused byRespiratory Syncytial Virus, malaria, schistosomiasis, andtrypanosomiasis.